Unit dose formulation of antidotes for factor xa inhibitors and methods of using the same

ABSTRACT

The present invention relates unit dose formulations of antidotes to anticoagulants targeting factor Xa. Disclosed herein are methods of stopping or preventing bleeding in a patient that is currently undergoing anticoagulant therapy with a factor Xa inhibitor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/836,536, filed Jul. 14, 2010, which application claims the benefitunder 35 U.S.C. §119(e) to U.S. Application No. 61/225,887, filed Jul.15, 2009. The entire content of each of these applications is herebyincorporated to the present disclosure by reference.

FIELD

The present invention relates to unit dose formulations of an antidotewhich reverses and/or neutralizes the factor Xa inhibitor. Specifically,the antidote may be factor Xa (fXa) derivatives having reduced orlacking intrinsic procoagulant activity but are also capable of bindingand/or neutralizing fXa inhibitors thereby acting as antidotes toanticoagulants targeting fXa. The invention is also related to methodsof using the particular dose of antidote.

BACKGROUND

Anticoagulants serve a need in the marketplace in treatment orprevention of undesired thrombosis in patients with a tendency to formblood clots, such as, for example, those patients having clottingdisorders, confined to periods of immobility or undergoing medicalsurgeries. One of the major limitations of anticoagulant therapy,however, is the bleeding risk associated with the treatments, andlimitations on the ability to rapidly reverse the anticoagulant activityin case of overdosing or if an urgent surgical procedure is required.Thus, specific and effective antidotes to all forms of anticoagulanttherapy are highly desirable. For safety considerations, it is alsoadvantageous to have an anticoagulant-antidote pair in the developmentof new anticoagulant drugs.

Currently available anticoagulant-antidote pairs forover-anticoagulation are heparin-protamine and warfarin-vitamin K. Freshfrozen plasma and recombinant factor VIIa (rfVIIa) have also been usedas non-specific antidotes in patients under low molecular weight heparintreatment, suffering from major trauma or severe hemorrhage. (Lauritzen,B. et al., Blood, 2005, 607A-608A.) Also reported are protaminefragments (U.S. Pat. No. 6,624,141) and small synthetic peptides (U.S.Pat. No. 6,200,955) as heparin or low molecular weight heparinantidotes; and thrombin muteins (U.S. Pat. No. 6,060,300) as antidotesfor thrombin inhibitor. Prothrombin intermediates and derivatives havebeen reported as antidotes to hirudin and synthetic thrombin inhibitors(U.S. Pat. Nos. 5,817,309 and 6,086,871).

One promising form of anticoagulant therapy targets factor Xa (fXa), andin fact, several direct fXa inhibitors are currently in different stagesof clinical development for use in anticoagulant therapy. One direct fXainhibitor Xarelto™ (rivaroxaban) has been approved for clinical use inthe European Union and Canada for the prevention of venousthromboembolism in orthopedic surgery patients. Many of these are smallmolecules. While these new fXa inhibitors show promise for treatment,specific and effective antidotes are still needed. In case ofover-anticoagulation or requirement for surgery in patients treated withthese fXa inhibitors, an agent may be required to substantiallyneutralize the administered fXa inhibitor or inhibitors and restorenormal hemostasis.

Currently available agents, such as recombinant factor VIIa (rfVIIa),are mechanistically limited and not specific for reversal of fXainhibitors and thus improved options for the clinician are highlydesirable. In human studies, rfVIIa has been used to reverse the effectof indirect antithrombin III dependent fXa inhibitors such asfondaparinux and idraparinux (Bijsterveld, N R et al., Circulation,2002, 106:2550-2554; Bijsterveld, N R et al., British J. of Haematology,2004(124): 653-658). The mechanism of action of factor VIIa (fVIIa) isto act with tissue factor to convert factor X (fX) present in bloodcirculation to fXa to restore normal hemostasis in patients. This modeof action necessarily dictates that the highest potential concentrationof fXa that could be attained to neutralize active site directed fXainhibitors is limited by the circulating plasma concentration of fX.Thus the potential of using rfVIIa to reverse the effect of direct fXainhibitors is mechanistically limited. Since the circulating plasmaconcentration of fX is 150 nanomolar (“nM”), the maximal amount of fXaproduced by this mode would be 150 nM. Reported therapeuticconcentrations of small molecule fXa inhibitors such as rivaroxaban havebeen higher (approximately 600 nM, Kubitza D, et al., Eur. J. Clin.Pharmacol., 2005, 61:873-880) than the potential amount of fXa generatedby rfVIIa. Use of rfVIIa for reversal of therapeutic or supratherapeuticlevels of anticoagulation by fXa inhibitor would therefore provideinadequate levels of efficacy. As shown in FIG. 4, using rfVIIa haslimited effect in neutralizing the anticoagulant activity of a factor Xainhibitor betrixaban (described below). Recombinant fVIIa showed a doseresponsive antidote activity from 50 nM to 100 nM, but the effectleveled off between 100 nM to 200 nM, indicating that its antidoteeffect is limited by factors other than its concentration. In all of therfVIIa concentrations tested, betrixaban still showed a dose responsiveinhibition of fXa, up to about 75% inhibition at a concentration of 250nM. This observation is consistent with fVIIa's proposed mechanism ofaction. This is also supported by studies showing that rfVIIa did notcompletely reverse the inhibitory effect of fondaparinux on theparameters of thrombin generation and prothrombin activation.(Gerotiafas, G T, et al., Thrombosis & Haemostasis 2204(91):531-537).

Exogenous active fXa cannot be administered directly to a subject in away similar to rfVIIa. Unlike rfVIIa, which has very low procoagulantactivity in the absence of its cofactor tissue factor, native fXa is apotent enzyme and has a potential risk of causing thrombosis. Thus, theuse of either rfVIIa or active fXa as an antidote to a fXa anticoagulanttherapy has disadvantages.

Antidotes employed in the formulations and methods of the invention aredescribed in U.S. Patent Application Publication 2009-0098119. Thispublication, and any publications, patents, patent applicationsmentioned herein, are hereby incorporated by reference in theirentirety.

Notwithstanding the disclosure of antidotes in the previously mentionedapplication, the dosing of the antidote is a critical component toassure patient safety.

SUMMARY

It has now been discovered that administration of modified derivativesof fXa proteins are useful as antidotes to anticoagulants targeting fXawhen provided in a certain dose. The modified derivatives of fXaproteins do not compete with fXa in assembling into the prothrombinasecomplex, but instead bind and/or substantially neutralize theanticoagulants, such as fXa inhibitors. The derivatives useful asantidotes are modified to reduce or remove intrinsic procoagulant andanticoagulant activities, while retaining the ability to bind to theinhibitors. It is contemplated that the derivatives of the invention mayinclude modifying the active site, or changing or removing the entireGla domain from fXa, or various combinations thereof. It is furthercontemplated that modification of the Gla domain reduces or removes theanticoagulant effect of the fXa derivative on normal hemostasis becausean active site modified full length fXa is known to be an anticoagulant.

In one embodiment, the invention is directed to a unit dose formulationcomprising a pharmaceutically acceptable carrier and a two chainpolypeptide comprising the amino acid sequence of SEQ ID NO. 13 or apolypeptide having at least 80% homology to SEQ ID NO. 13, in an amountfrom about 10 milligrams to about 2 grams. In some embodiments, theamount is from about 100 milligrams to about 1.5 grams or from about 200milligrams to about 1 gram or from about 400 milligrams to about 900milligrams.

In certain embodiments, the amount of the polypeptide is effective inneutralizing a factor Xa inhibitor by about 20%, 50%, 75%, 90%, 95%, 99%or about 100%.

In another embodiment, the invention is directed to a unit doseformulation for administration to a subject undergoing anticoagulanttherapy with a factor Xa inhibitor, said formulation comprising apharmaceutically acceptable carrier and a neutralizing amount of a twochain polypeptide comprising the amino acid sequence of SEQ ID NO. 13 ora polypeptide having at least 80% homology to SEQ ID NO. 13, such thatthe neutralizing amount is at least about a 1:1 fold molar ratio ofcirculating concentration of polypeptide over circulating concentrationof the factor Xa inhibitor for a period of at least about 30 minutes. Inone embodiment, this molar ratio relates to betrixaban-inducedanticoagulation. In other embodiments the molar ratio is about 1:1 orabout 2:1 and in still other embodiments, the ratio is about 4:1 orhigher.

In some embodiments, the carrier is saline. In some embodiments thecarrier is sterile saline. In other embodiments, the formulation has aconcentration of from about 0.2 to about 10 milligrams of polypeptideper milliliter of saline. In other embodiments, the concentration isfrom about 2 to about 6 milligrams of polypeptide per milliliter ofsaline or about 2 milligrams of polypeptide per milliliter of saline.

In certain embodiments, the polypeptide is lyophilized.

In another embodiment, the invention is directed to a method ofselectively binding and inhibiting an exogenously administered factor Xainhibitor in a subject undergoing anticoagulant therapy with a factor Xainhibitor comprising administering to the subject a unit doseformulation of the invention.

In still another embodiment, the invention is directed to a method ofpreventing, reducing, or ceasing bleeding in a subject undergoinganticoagulant therapy with a factor Xa inhibitor comprisingadministering to the subject a unit dose formulation of the invention.

In still another embodiment, the invention is directed to a method forcorrecting fXa inhibitor dependent pharmacodynamic or surrogate markersin a patient undergoing anticoagulant therapy with a factor Xa inhibitorcomprising administering to the subject a unit dose formulation of theinvention.

In the methods of the invention, the formulation may be administered viaeither intravenous administration by bolus or a combination of bolus andinfusion or by subcutaneous administration. Subcutaneous dosing of humanclotting factors has been reported in the literature. See, McCarthy K.,et al., Thromb. Haemost., 2002, 87(5): 824-30; Gerrard A J, et al., Br.J. Haematol., 1992, 81(4): 610-3; Miekka S I et al., Haemophilia, 1998,4(4), 436-42. In certain embodiments, about 10 to about 20% of theformulation is administered as a bolus and the remaining formulation isinfused over a period until bleeding has substantially ceased. It iscontemplated that the infusion can be administered for about 6 hours, orabout 6 to about 12 hours, or about 12 to about 24 hours or 48 hours.

In another aspect, the modified factor Xa protein is co-administeredwith an agent capable of extending the plasma half life (or circulatinghalf life) of the factor Xa derivative. In yet another aspect, theantidote is conjugated with a moiety to extend its plasma half-life.

In another aspect, this invention provides a kit comprising a fXainhibitor for anticoagulant use and a fXa inhibitor antidote (or factorXa derivative) for use when substantial neutralization of the fXainhibitor's anticoagulant activity is needed. When the antidote isprovided in lyophilized form, the kit optionally further comprises avial of sterile saline.

Further provided herein is a peptide conjugate comprising a carriercovalently or non-covalently linked to a polypeptide just described. Thecarrier can be a liposome, a micelle, a pharmaceutically acceptablepolymer, or a pharmaceutically acceptable carrier.

Additional embodiments of the invention may be found throughout theremainder of the specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically the domain structure of human factor X (SEQID NO. 1) shown in Table 13 as reported in Leytus et al., Biochem.,1986, 25, 5098-5102. SEQ ID NO. 1 is the amino acid sequence of human fXcoded by the nucleotide sequence of human fX (SEQ ID NO. 2) as shown inTable 14 known in the prior art. For example, the translated amino acidsequence is reported in Leytus et al., Biochem., 1986, 25, 5098-5102 andcan be found in GenBank, “NM_(—)000504” at<http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=89142731>.The amino acid numbering in this sequence is based on fX sequence. HumanfX precursor (SEQ ID NO. 1) contains a prepro-leader sequence (aminoacids 1 to 40 of SEQ ID NO. 1) followed by sequences corresponding tothe fX light chain (LC) (amino acids 41 to 179 of SEQ ID NO. 1), the RKR(SEQ ID NO. 16) triplet (amino acids 180 to 182 of SEQ ID NO. 1) whichis removed during fX secretion, and the fX heavy chain (amino acids 183to 488 of SEQ ID NO. 1) containing the activation peptide (AP) (aminoacids 183 to 234 of SEQ ID NO. 1) and the catalytic domain (amino acids235 to 488 of SEQ ID NO. 1).

FIG. 2 (SEQ ID NO. 3) shows the amino acid sequence of mature humanfactor X. The amino acid numbering in this figure is based on mature fXsequence starting from the N-terminal of fX light chain. Factor Xcirculates in plasma as a two-chain molecule linked by a disulfide bond.The light chain (LC) has 139 amino acid (amino acids 41 through 179 ofSEQ ID NO. 1) residues and contains the γ-carboxyglutamic acid(Gla)-rich domain (amino acids 1-45 of SEQ ID NO. 3), including a shortaromatic stack (AS) (amino acids 40-45 of SEQ ID NO. 3), followed by twoepidermal growth factor (EGF)-like domains (EGF1: amino acids 46-84,EGF2: amino acids 85-128 of SEQ ID NO. 3). The heavy chain (HC) has 306amino acids and contains a 52 amino acids activation peptide (AP: aminoacids 143-194 of SEQ ID NO. 3) followed by the catalytic domain (aminoacids 195-448 of SEQ ID NO. 3). The catalytic triad equivalents toH57-D102-5195 in chymotrypsin numbering are located at His236, Asp282,and Ser379 in fX sequence and are underlined (amino acids 236, 282 and379 of SEQ ID NO. 3).

FIG. 3 shows schematically the domain structure of mature human factor Xshown in FIG. 2. The amino acid numbering in this figure is based onmature fX sequence. The cleavage sites for chymotrypsin digestion toremove the Gla-domain containing fragment (amino acid 1-44 of SEQ ID NO.3) and fX activation to remove the activation peptide are highlighted.Chymotrypic digestion of fXa results in a Gla-domainless fXa lacking the1-44 amino acid residues (SEQ ID NO. 4).

FIG. 4 shows the effect of varying concentrations of rfVIIa in thepresence of tissue factor on the anticoagulant activity of a fXainhibitor betrixaban (described below) in a thrombin generation(expressed as relative fluorescence units (RFU) assay (as described inExample 2)) using PPP prepared samples. The data show that a combinationof rfVIIa and tissue factor was unable to completely neutralize theanticoagulant activity of a fXa inhibitor, betrixaban, in concentrationsup to 250 nM.

FIG. 5 shows that anhydro-fXa with its Gla-domain intact reverses fXainhibition by betrixaban in a purified system containing active fXa andbetrixaban (open circle), while anhydro-fXa alone has negligibleprocoagulant activity (open triangle) compared with active fXa. FXachromogenic activity was normalized to active fXa in the absence of anyinhibitor (open square). This is more thoroughly described in Example 4.The data show that anhydro-fXa is inactive toward fXa substrate yetretains the fXa inhibitor binding ability.

FIG. 6 shows that the anhydro-fXa with intact Gla domain in FIG. 5 is apotent inhibitor in plasma thrombin generation (expressed as relativefluorescence units (RFU)) assay using PPP prepared samples (as describedin Example 2). It almost completely inhibited thrombin generation atabout 115 nM. The data show that anhydro-fXa without modification of theGla-domain is not suitable for use as a fXa inhibitor antidote.

FIG. 7 shows the comparison of the clotting activity of active fXa in a96-well plate format before chymotrypsin digestion, and after 15 minutesand 30 minutes of chymotrypsin digestion. As shown in this figure,clotting time (change of OD405) was significantly delayed after the fXahad been digested by chymotrypsin for 15 minutes and no clotting wasobserved for up to 20 minutes when the fXa was digested for 30 minutes.This result was also used to establish conditions for chymotrypsindigestion of anhydro-fXa because it has no activity that can bemonitored during digestion. This is more thoroughly described in Example1.

FIG. 8 shows the binding affinity of des-Gla anhydro-fXa to a factor Xainhibitor betrixaban as described in Example 4. The data show thatdes-Gla anhydro-fXa, prepared by chymotryptic digestion of anhydro-fXato remove the Gla-domain containing fragment (residues 1-44), is able tobind betrixaban with similar affinity as native fXa (fXa: Ki=0.12 nM,des-Gla anhydro-fXa: Kd=0.32 nM).

FIG. 9 shows reversal of the anticoagulant activity of varyingconcentrations of betrixaban by addition of a concentrate of 680 nM ofthe antidote (des-Gla anhydro-fXa) in a thrombin generation assay ofExample 2 using PPP prepared samples. At the concentration of 680 nM,des-Gla anhydro-fXa was able to produce substantially completerestoration of fXa activity.

FIG. 10 shows reversal of the anticoagulant activity of 250 nM ofbetrixaban by varying concentrations of the antidote (des-Glaanhydro-fXa) in clotting prolongation assays with PPP prepared samplesusing aPTT reagent in a 96-well plate format (as described in Example3). The data show that clotting time was comparable to that of controlplatelet poor plasma when about 608 nM of the antidote was used toneutralize 250 nM of the fXa inhibitor betrixaban.

FIG. 11 shows the effect on the anticoagulant activity of enoxaparin(0.3125-1.25 U/mL) by 563 nM of the antidote (des-Gla anhydro-fXa) inclotting prolongation assays with PPP prepared samples using aPTTreagent in a 96-well plate format, expressed as fold changes afternormalization. The assay protocol is described in Example 3. The datashow that addition of 563 nM of the antidote significantly neutralizedthe activity of a low molecular weight heparin enoxaparin.

FIG. 12 shows the effect of the antidote, des-Gla anhydro-fXa, on theactivity of thrombin (5 nM) and its inhibition by 50 nM of argatroban, aspecific thrombin inhibitor, in a chromogenic assay. As expected, theantidote of fXa inhibitor does not detectably affect either thrombinactivity or its inhibition by the specific inhibitor argatroban atconcentrations up to 538 nM. This is more thoroughly described inExample 14.

FIG. 13 shows the effect on the anticoagulant activity of 400 nMbetrixaban by varying concentrations of the antidote, des-Glaanhydro-fXa, in an aPTT assay using a standard coagulation timer. Theassay protocol is described in Example 3. The data shows that theantidote of fXa inhibitor substantially reverses the inhibition of fXaby 400 nM of betrixaban. The EC₅₀ of the antidote was estimated to beabout 656 nM with 400 nM betrixaban.

FIG. 14 shows the map of the DNA construct for expression of the fXatriple mutant (SEQ ID NO. 12) in CHO cells. Plasmid DNA was linearizedand transfected into CHO dhfr(−) cells. Cells were selected usingtetrahydrofolate (HT) deficient media plus methotrexate (MTX). Stableclones were screened for high protein expression by ELISA. The fXatriple mutant was produced in serum free medium and purified bycombination of ion exchange and affinity columns. The numbering in themap was based on polynucleotide sequence encoding human fX SEQ ID NO. 1.For example, an alanine mutation at the active site 5419 (SEQ ID NO. 1)is equivalent to the mutation at 5379 (SEQ ID NO. 3) of mature human fXdiscussed throughout the application and more particularly, Example 7.

FIGS. 15A-C show SDS-PAGE and Western blot of purified r-Antidote usingmonoclonal antibodies recognizing human fX heavy chain and light chain,respectively.

FIG. 15A shows a Western blot of purified r-Antidote by ion exchange andaffinity purification. Upon reduction of the disulfide bond whichconnects the light and heavy chains, the r-Antidote heavy chain migratesat expected molecular weight similar to that of plasma derived fXa.Deletion of 6-39 aa in the Gla-domain of fXa mutant results in a lowermolecular weight band of the r-Antidote light chain compared to normalFXa.

FIGS. 15B and 15C show a SDS-PAGE and Western blot of purifiedr-Antidote by ion exchange and affinity purification followed by sizeexclusion chromatography.

FIG. 16 shows betrixaban plasma level in mice (n=7-10 per group) afteroral administration of betrixaban alone (15 mg/kg), or betrixaban (15mg/kg) followed by intravenous injection (300 μg, IV) of plasma derivedantidote (pd-Antidote) prepared according to Example 1. pd-Antidote wasadministered 5 minutes prior to the 1.5 hr. time point, and mouse bloodsamples (0.5 mL) were taken at 1.5, 2.0, and 4.0 hrs following oraladministration of betrixaban. Whole blood INR, betrixaban and antidoteplasma levels were analyzed. Betrixaban level (Mean±SEM) in mouse plasmawas plotted as a function of time for mice after 15 mg/kg (open square)and 15 mg/kg followed by antidote injection (open circle). The PK-PDcorrelation of antidote treated group at 1.5 hr time point (5 min afterantidote injection) was summarized in Table 1. Single injection of theantidote resulted in >50% reduction of functional betrixaban based onINR measurements. This is more thoroughly described in Example 8.

FIGS. 17A-B show the results of a mouse experiment with purifiedr-Antidote (n=4-10 per group). Betrixaban level in mouse plasma (FIG.17A) and whole blood INR (FIG. 17B) were compared after oraladministration of betrixaban alone (15 mg/kg) or betrixaban (15 mg/kg)followed by intravenous injection (300 μg) of r-antidote. Mean valuesfor each treated group were indicated. As summarized in Table 2, singleIV injection of the r-antidote resulted in >50% correction of ex vivowhole blood INR, justifying effective neutralization of fXa inhibitorsby the antidote via a single or multiple injections or other regimes.These results demonstrate that the fXa variants of this invention havepotential of acting as universal antidotes to reverse the anticoagulanteffect of fXa inhibitors in patients with bleeding or other medicalemergencies. This is more thoroughly described in Example 8.

FIG. 18 shows r-Antidote reversal of the inhibitory effect of enoxaparinin a 96-well turbidity change clotting assay. The results areessentially similar to pd-Antidote (FIG. 11) indicating both fXaderivatives have comparable functional antidote activity. 508 nMr-Antidote substantially corrected (>75%) the inhibitory effect of 1.25U/mL enoxaparin. The assay protocol is presented in Example 11.

FIG. 19 shows r-Antidote reversal of the inhibitory effect of lowmolecular weight heparin (LMWH) as tested in human plasma clottingassay. Both FIGS. 18 and 19 are discussed in Example 11.

FIG. 20 shows the r-Antidote reversal of the anticoagulation effect ofrivaroxaban. This is more thoroughly discussed in Example 12.

FIG. 21 shows the alignment of the polynucleotide sequence andtranslated polypeptide sequence of r-Antidote.

FIGS. 22A-B show the results of a mouse experiment with a single IVinjection (1 injection) or two injections (2 injections) of ther-antidote (n=5 per group, 312 ug/200 ul r-Antidote). Betrixaban levelin plasma (FIG. 22A) were compared after oral administration ofbetrixaban (15 mg/kg) followed by intravenous injection of vehicle orr-Antidote (see Example 8 for details). As shown in FIG. 22A, a singleIV injection of r-Antidote increased betrixaban level in plasma by morethan 8 fold compared to vehicle control (control_(—)1), indicating theability of the antidote to effectively bind betrixaban in vivo. A secondinjection of the antidote further increased betrixaban level by lessthan 2 fold compared to the single injection, indicating limiting amountof betrixaban in mouse blood and reversal of its anticoagulant effect bythe antidote. FIG. 22B demonstrates that measured INR decreases as theratio of antidote/betrixaban increases in mouse plasma following singleand double injections of the antidote.

FIGS. 23A-C show the reversal of fXa inhibition by rivaroxaban (A),betrixaban (B) and apixaban (C) using the r-Antidote. Curve fit and dataanalysis was carried out by using Dynafit and Graphpad Prism software(Example 15).

FIG. 24 shows r-Antidote reversal of prolongation of PT by rivaroxabanin human plasma (Example 16).

FIG. 25 shows prolongation of PT by apixaban and reversal of itsanticoagulant effects by addition of r-Antidote (Example 16).

FIG. 26 shows r-Antidote reversal of anticoagulation effect ofenoxaparin (Example 17).

FIG. 27 shows a dose responsive reversal of rivaroxaban-inducedanticoagulation by IV administration of r-Antidote in rats (Example 18).

FIG. 28 shows a dose responsive reduction of free (unbound) fraction ofrivaroxaban upon dosing of r-antidote into anticoagulated rats (Example19).

FIGS. 29 A and B show sustained reversal of rivaroxaban-inducedanticoagulation by IV administration of r-Antidote in rats as measuredby whole blood INR and PT ratio (Example 20).

FIG. 30 shows dose ranging study of enoxaparin in anesthetized rats(Example 21).

FIG. 31 shows sustained reversal of enoxaparin-induced anticoagulationby IV administration of r-antidote and protamine sulfate in rats asmeasured by activated partial thromboplastin times (Example 21).

FIG. 32 shows sustained reversal of betrixaban-induced anticoagulationby administration of r-Antidote (Example 22).

FIG. 33 shows plasma concentration-time profile of r-Antidote inSprague-Dawley rat following 1 mg intravenous dose (Example 23).

FIG. 34 shows plasma concentration-time profile of r-Antidote in Rhesusmonkey following 10 mg intravenous dose (Example 24).

FIGS. 35A and 35B show simulated time course profile of neutralizationof rivaroxaban activity by administration of r-Antidote. In FIG. 35A, a20 mg dose of rivaroxaban is reversed by a 400 mg dose of r-Antidote(bolus dosing) while assuming a T_(1/2) of 3 hours for the r-Antidote.In FIG. 35B, a 20 mg dose of rivaroxaban is reversed using a 900 mg doseof r-Antidote (bolus plus 6 hour infusion) while assuming a T_(1/2) of 1hour for the r-Antidote (Example 25).

FIGS. 36A and 36B show simulated time course profile of neutralizationof betrixaban activity by r-Antidote. In FIG. 36A, a 80 mg dose ofbetrixaban is reversed by a 400 mg dose of r-Antidote (bolus dosing)while assuming a T_(1/2) of 3 hours for the r-Antidote. In FIG. 36B a 80mg dose of betrixaban is reversed using a 900 mg dose of r-Antidote(bolus plus 6 hour infusion) while assuming a T_(1/2) of 1 hour for ther-Antidote (Example 26).

FIG. 37 shows the effect of r-Antidote on reversal of anticoagulation byrivaroxaban (Example 27).

FIG. 38 shows reversal of blood loss due to enoxaparin anticoagulationby administration of the r-Antidote in a rat (Example 28). Blood lossesfor individual animals in each treated group were shown in FIG. 41.

FIG. 39 shows the reversal of blood loss due to fondaparinuxanticoagulation by administration of the r-Antidote in a rat (Example28). Blood losses for individual animals in each treated group wereshown in FIG. 43.

FIG. 40 shows the reversal of anticoagulation due to enoxaparinanticoagulation by administration of the r-Antidote. The anticoagulationwas measured by plasma anti-fXa units (Example 29).

FIG. 41 shows the dose responsive mitigation of blood loss due toenoxaparin anticoagulation by administration of the r-Antidote in a rat(Example 28).

FIGS. 42 a-c show the correlation of blood loss measured in the rat tailtransaction model (example 28) and enoxaparin concentrations as measuredby anti-fXa units (Example 29).

FIG. 42 a shows a steep increase in blood loss as enoxaparinconcentrations increased to greater than 1.5 U/mL measured by anti-fXaunits assay.

FIG. 42 b shows a correlation analysis with an r² value of 0.799 betweenblood loss and r-antidote concentrations.

FIG. 42 c shows a correlation analysis with an r² value of 0.689 betweenanti-fXa units and r-antidote concentrations.

FIG. 43 shows the reversal of blood loss due to fondaparinuxanticoagulation with the r-antidote but not protamine in the rat tailtransaction model (Example 28).

FIG. 44 shows the reversal of anticoagulation due to fondaparinux asmeasured by anti-fXa activity assay.

DETAILED DESCRIPTION I. Definitions

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of tissue culture, immunology,molecular biology, microbiology, cell biology and recombinant DNA, whichare within the skill of the art. See, e.g., Sambrook and Russell eds.(2001) Molecular Cloning: A Laboratory Manual, 3^(rd) edition; theseries Ausubel et al. eds. (2007) Current Protocols in MolecularBiology; the series Methods in Enzymology (Academic Press, Inc., N.Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press atOxford University Press); MacPherson et al. (1995) PCR 2: A PracticalApproach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual;Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique,5^(th) edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Pat. No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization;Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds.(1984) Transcription and Translation; Immobilized Cells and Enzymes (IRLPress (1986)); Perbal (1984) A Practical Guide to Molecular Cloning;Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells(Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer andExpression in Mammalian Cells; Mayer and Walker eds. (1987)Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Herzenberg et al. eds (1996) Weir's Handbook of ExperimentalImmunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^(rd)edition (Cold Spring Harbor Laboratory Press (2002)).

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a pharmaceutically acceptable carrier”includes a plurality of pharmaceutically acceptable carriers, includingmixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

A “subject” of diagnosis or treatment is a cell or a mammal, including ahuman. Non-human animals subject to diagnosis or treatment include, forexample, murine, such as rats, mice, canine, such as dogs, leporids,such as rabbits, livestock, sport animals, and pets.

The term “protein” and “polypeptide” are used interchangeably and intheir broadest sense to refer to a compound of two or more subunit aminoacids, amino acid analogs or peptidomimetics. The subunits may be linkedby peptide bonds. In another embodiment, the subunit may be linked byother bonds, e.g., ester, ether, amino, etc. A protein or peptide mustcontain at least two amino acids and no limitation is placed on themaximum number of amino acids which may comprise a protein's orpeptide's sequence. As used herein the term “amino acid” refers toeither natural and/or unnatural or synthetic amino acids, includingglycine and both the D and L optical isomers, amino acid analogs andpeptidomimetics. Single letter and three letter abbreviations of thenaturally occurring amino acids are listed below. A peptide of three ormore amino acids is commonly called an oligopeptide if the peptide chainis short. If the peptide chain is long, the peptide is commonly called apolypeptide or a protein.

1-Letter 3-Letter Amino Acid Y Tyr L-tyrosine G Gly L-glycine F PheL-phenylalanine M Met L-methionine A Ala L-alanine S Ser L-serine I IleL-isoleucine L Leu L-leucine T Thr L-threonine V Val L-valine P ProL-proline K Lys L-lysine H His L-histidine Q Gln L-glutamine E GluL-glutamic acid W Trp L-tryptohan R Arg L-arginine D Asp L-aspartic acidN Asn L-asparagine C Cys L-cysteine

“Factor Xa” or “fXa” or “fXa protein” refers to a serine protease in theblood coagulation pathway, which is produced from the inactive factor X(fX). Factor Xa is activated by either factor IXa with its cofactor,factor VIIIa, in a complex known as intrinsic Xase, or factor VIIa withits cofactor, tissue factor, in a complex known as extrinsic Xase. fXaforms a membrane-bound prothrombinase complex with factor Va and is theactive component in the prothrombinase complex that catalyzes theconversion of prothrombin to thrombin. Thrombin is the enzyme thatcatalyzes the conversion of fibrinogen to fibrin, which ultimately leadsto blood clot formation. Thus, the biological activity of fXa issometimes referred to as “procoagulant activity” herein.

The nucleotide sequence coding human factor X (“fX”) can be found inGenBank, “NM_(—)000504” at<http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=89142731>,and is listed in FIG. 1 b and SEQ ID No. 2. The corresponding amino acidsequence and domain structure of fX are described in Leytus et al.,Biochemistry, 1986, 25:5098-5102. The domain structure of mature fX isalso described in Venkateswarlu, D. et al., Biophysical Journal, 2002,82:1190-1206. Upon catalytic cleavage of the first 52 residues (aminoacids 143 to 194 of SEQ ID NO. 3) of the heavy chain, fX is activated tofXa (SEQ ID NO. 6). FXa contains a light chain (SEQ ID NO. 8) and aheavy chain (SEQ ID NO. 9). The first 45 amino acid residues (residues1-45 of SEQ ID NO. 6) of the light chain is called the Gla domainbecause it contains 11 post-translationally modified γ-carboxyglutamicacid residues (Gla). It also contains a short (6 amino acid residues)aromatic stack sequence (residues 40-45 of SEQ ID NO. 6). Chymotrypsindigestion selectively removes the 1-44 residues resulting inGla-domainless fXa (SEQ ID NO. 4). The serine protease catalytic domainof fXa locates at the C-terminal heavy chain. The heavy chain of fXa ishighly homologous to other serine proteases such as thrombin, trypsin,and activated protein C.

The domain structure of mature factor X may be found in Venkateswarlu D.et al., Biophysical J., 2002, 82, 1190-1206, which is herebyincorporated by reference in its entirety. The amino acid numbering inthis figure is the same as in FIG. 3. The tripeptide ofArg140-Lys141-Arg142 (the RKR (SEQ ID NO. 16) triplet as shown inFIG. 1) that connects the light chain to the activation peptide is notshown because the form that lacks the tripeptide is predominant incirculation blood plasma. Individual domains are shown in boxes. Thisincludes amino acids 1-45 in FIG. 2 (SEQ ID NO. 3). Functionallyimportant catalytic residues are circled, and “γ” represents Gla(γ-carboxyglutamic acid) residue.

“Native fXa” or “wild-type fXa” refers to the fXa naturally present inplasma or being isolated in its original, unmodified form, whichprocesses the biological activity of activating prothrombin thereforepromoting formation of blood clot. The term includes naturally occurringpolypeptides isolated from tissue samples as well as recombinantlyproduced fXa. “Active fXa” refers to fXa having the biological activityof activating prothrombin. “Active fXa” may be a native fXa or modifiedfXa that retains procoagulant activity.

“fXa Derivatives” or “modified fXa” or “derivatives of a factor Xaprotein” refers to fXa proteins that have been modified such that theybind, either directly or indirectly, to a factor Xa inhibitor and do notassemble into the prothrombinase complex. Structurally, the derivativesare modified to provide either no procoagulant activity or reducedprocoagulant activity. “Procoagulant activity” is referred to herein asan agent's ability to cause blood coagulation or clot formation. Reducedprocoagulant activity means that the procoagulant activity has beenreduced by at least about 50%, or more than about 90%, or more thanabout 95% as compared to wild-type fXa during the same time period. Forexample, recombinant fX-S395A essentially has no procoagulant activityas measured by in vitro assays, such as fXa activity assays.

The derivatives have either modified active sites or modified Gladomains or both. Additional modifications are also contemplated. It iscontemplated that such modifications may be made in one or more of thefollowing ways: deletion of one or more of the amino acid from thesequence, substitution of one or more amino acid residues with one ormore different amino acid residues, and/or manipulation of one or moreamino acid side chains or its “C” or “N” terminals.

The term “active site” refers to the part of an enzyme or antibody wherea chemical reaction occurs. A “modified active site” is an active sitethat has been modified structurally to provide the active site withincreased or decreased chemical reactivity or specificity. Examples ofactive sites include, but are not limited to, the catalytic domain ofhuman factor X comprising the 235-488 amino acid residues (FIG. 1), andthe catalytic domain of human factor Xa comprising the 195-448 aminoacid residues (FIGS. 2 and 3). Examples of modified active site include,but are not limited to, the catalytic domain of human factor Xacomprising 195-448 amino acid residues in SEQ ID NOS. 10, 11, 12, 13, or15 with at least one amino acid substitution at position Arg306, Glu310,Arg347, Lys351, Lys414, or Arg424.

As stated above, the derivatives of the invention may have modified Gladomains or have the entire Gla domain removed. Examples of fXaderivatives suitable as antidotes in the methods of this invention areGla-domainless fXa (SEQ ID NOS. 4 or 5), Gla-deficient fXa (SEQ ID NO. 7with modifications described herein), fXa with modifications at thecatalytic site (SEQ ID NOS. 10 or 11), and fXa with modifications at thesites known to be important for fV/fVa interaction or fVIII/fVIIIainteraction (SEQ ID NOS. 4, 5, 7, 10, or 11 with at least one amino acidsubstitution at position Arg306, Glu310, Arg347, Lys351, Lys414 orArg424), as described in detail herein. Further examples of the fXaderivatives contemplated by this invention are provided below.

“Gla-domainless fXa” or “des-Gla fXa” refers to fXa that does not have aGla-domain and encompasses fXa derivatives bearing other modification(s)in addition to the removal of the Gla-domain. Examples of Gla-domainlessfXa in this invention include, but are not limited to, fXa derivativelacking the 1-39 amino acid residues of SEQ ID NO. 3; fXa derivativelacking the 6-39 amino acid residues of SEQ ID NO. 3, corresponding to afXa mutant expressed in CHO cells described in more details below (SEQID NO. 12, Table 24); fXa derivative lacking the 1-44 amino acidresidues of SEQ ID NO. 3, corresponding to des-Gla fXa afterchymotryptic digestion of human fXa (SEQ ID NO. 4, FIG. 3); and fXaderivative lacking the entire 1-45 Gla-domain residues of SEQ ID NO. 3as described in Padmanabhan et al., Journal Mol. Biol., 1993,232:947-966 (SEQ ID NO 5). Other examples include des-Gla anhydro fXa(SEQ ID NO. 10, Table 22) and des-Gla fXa-S379A (SEQ ID NO. 11, Table23).

In some embodiments, the des-Gla fXa comprises at least amino acidresidues 40 to 448 of SEQ ID NO. 3 or an equivalent thereof. In someembodiment, the des-Gla fXa comprises at least amino acid residues 45 to488 (SEQ ID NO. 4) or 46 to 488 (SEQ ID NO. 5) of SEQ ID NO. 3 orequivalents thereof.

In some embodiment, the des-Gla fXa comprises at least amino acidresidues 40 to 139 and 195 to 448 of SEQ ID NO. 3 or equivalentsthereof. In some embodiment, the des-Gla fXa comprises at least aminoacid residues 45 to 139 and 195 to 448 of SEQ ID NO. 3 or equivalentsthereof. In another embodiment, the des-Gla fXa comprises at least aminoacid residues 46 to 139 and 195 to 448 of SEQ ID NO. 3 or equivalentsthereof.

“Gla-deficient fXa” refers to fXa with reduced number of free side chainγ-carboxyl groups in its Gla-domain. Like Gla-domainless fXa,Gla-deficient fXa can also bear other modifications. Gla-deficient fXaincludes uncarboxylated, undercarboxylated and decarboxylated fXa.“Uncarboxylated fXa” or “decarboxylated fXa” refers to fXa derivativesthat do not have the γ-carboxy groups of the γ-carboxyglutamic acidresidues of the Gla domain, such as fXa having all of its Gla domainγ-carboxyglutamic acid replaced by different amino acids, or fXa havingall of its side chain γ-carboxyl removed or masked by means such asamination, esterification, etc. For recombinantly expressed protein,uncarboxylated fXa is, sometimes, also called non-carboxylated fXa.“Undercarboxylated fXa” refers to fXa derivatives having reduced numberof γ-carboxy groups in the Gla domain as compared with wild-type fXa,such as fXa having one or more but not all of its Gla domainγ-carboxyglutamic acids replaced by one or more different amino acids,or fXa having at least one but not all of its side chain γ-carboxylremoved or masked by means such as amination and esterification, etc.

The domain structure of human Gla-domainless factor Xa may be found inPadmanabhan et al., J. Mol. Biol., 1993, 232, 947-966, which is herebyincorporated by reference in its entirety. The numbering of the aminoacid is based on topological equivalences with chymotrypsin, where, forexample, Ser195 corresponds to Ser379 in FIG. 2 when the human mature fXnumbering is used. Insertions are indicated with letters, and deletionsare indicated by 2 successive numberings. 300 are added to light chainnumbering to differentiate from the heavy chain numbering. β363 isβ-hydroxy aspartate. Slashes indicate proteolytic cleavages observed incrystallized material. The sequence of Gla-domainless fXa lacking the1-45 amino acid residues based mature fX (SEQ ID NO. 3) is listed in SEQID NO. 5.

In one embodiment, the fXa derivative may lack a light chain of fXa butstill contains a serine protease catalytic domain present in the heavychain. In addition chimeras with other serine protease catalytic domainmay be used to make substitutions in the heavy chain.

“pd-Antidote” or “plasma-derived antidote” refers to the des-Gla anhydrofXa derivative and has the amino acid residues of SEQ ID NO. 10.

“r-Antidote” or “recombinant antidote” refers to a fXa derivativelacking the 6-39 amino acid residues of SEQ ID NO. 3, corresponding to afXa mutant expressed in CHO cells and after removal of the linkerdescribed in more details below (SEQ ID NO. 13, Table 25).

“Anticoagulant agents” or “anticoagulants” are agents that inhibit bloodclot formation. Examples of anticoagulant agents include, but are notlimited to, specific inhibitors of thrombin, factor IXa, factor Xa,factor XIa, factor XIIa or factor VIIa, heparin and derivatives, vitaminK antagonists, and anti-tissue factor antibodies. Examples of specificinhibitors of thrombin include hirudin, bivalirudin (Angiomax®),argatroban and lepirudin (Refludan®). Examples of heparin andderivatives include unfractionated heparin (UFH), low molecular weightheparin (LMWH), such as enoxaparin (enoxaparine, Clexane®, Lovenox®,etc.), dalteparin (Fragmin®), nadroparin (Fraxiparin, Fraxiparine,etc.), tinzaparin (Innohep), ardeparin (Normiflo), certoparin(sandoparin, embolex, etc.) and danaparoid (Orgaran®); and syntheticpentasaccharide, such as fondaparinux (Arixtra®), idraparinux,idradbiotaparinux, and biotinylated idraparinux. Examples of vitamin Kantagonists include warfarin (Coumadin®), phenocoumarol, acenocoumarol(Sintrom®), clorindione, dicumarol, diphenadione, ethyl biscoumacetate,phenprocoumon, phenindione, and tioclomarol. In one embodiment, theanticoagulant is an inhibitor of factor Xa. In one embodiment, theanticoagulant is betrixaban.

“Anticoagulant therapy” refers to a therapeutic regime that isadministered to a patient to prevent undesired blood clots orthrombosis. An anticoagulant therapy comprises administering one or acombination of two or more anticoagulant agents or other agents at adosage and schedule suitable for treating or preventing the undesiredblood clots or thrombosis in the patient.

The term “factor Xa inhibitors” or “inhibitors of factor Xa” refers tocompounds that can inhibit, either directly or indirectly, thecoagulation factor Xa's activity of catalyzing conversion of prothrombinto thrombin in vitro and/or in vivo. Examples of known fXa inhibitorsinclude, without limitation, edoxaban, fondaparinux, idraparinux,biotinylated idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissuefactor pathway inhibitor, DX-9065a (as described in, e.g., Herbert, J.M., et al., J Pharmacol Exp Ther. 1996 276(3):1030-8), YM-60828 (asdescribed in, e.g., Taniuchi, Y., et al., Thromb Haemost. 199879(3):543-8), YM-150 (as described in, e.g., Eriksson, B. I. et. al,Blood 2005; 106(11), Abstract 1865), apixaban, rivaroxaban, PD-348292(as described in, e.g., Pipeline Insight: Antithrombotics—Reaching theUntreated Prophylaxis Market, 2007), otamixaban, razaxaban (DPC906), BAY59-7939 (as described in, e.g., Turpie, A. G., et al., J. Thromb.Haemost. 2005, 3(11):2479-86), edoxaban (as described in, e.g., Hylek EM, Curr Opin Invest Drugs 2007 8(9):778-783), LY517717 (as described in,e.g., Agnelli, G., et al., J. Thromb. Haemost. 2007 5(4):746-53),GSK913893, betrixaban (as described below) and derivatives thereof. Lowmolecular weight heparin (“LMWH”) is also considered a factor Xainhibitor.

In one embodiment, the factor Xa inhibitor is selected from betrixaban,rivaroxaban, apixaban, edoxaban, LMWH, and combinations thereof.

The term “betrixaban” refers to the compound“[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide”or pharmaceutically acceptable salts thereof.“[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide”refers to the compound having the following structure:

or a tautomer or pharmaceutically acceptable salt thereof.

Betrixaban is described in U.S. Pat. Nos. 6,376,515; 6,835,739; and7,598,276 the contents of which are incorporated herein by reference.Betrixaban is known to be a specific inhibitor of factor Xa.

As used herein, the term “antidote” or “antidote to a factor Xainhibitor” refers to molecules, such as derivatives of fXa, which cansubstantially neutralize or reverse the coagulation inhibitory activityof a fXa inhibitor by competing with active fXa to bind with availablefXa inhibitors. Examples of the antidotes of this invention are fXaderivatives with reduced phospholipid membrane binding, such as des-GlafXa or Gla-deficient fXa, and fXa derivatives with reduced catalyticactivity, such as the active site modified fXa derivatives, andderivatives with reduced interaction with fV/Va, or fVIII/fVIIIa.Examples of antidotes of the invention with reduced membrane binding andreduced catalytic activity include, but are not limited to, des-Glaanhydro-fXa by chymotryptic digestion of anhydro-fXa (as described inExample 1); des-Gla fXa-S379A (S195A in chymotrypsin numbering) bymutagenesis (as described in Example 6).

Other examples of antidotes of the invention include proteins orpolypeptides containing serine protease catalytic domains which possesssufficient structural similarity to fXa catalytic domain and aretherefore capable of binding small molecule fXa inhibitors. Examplesinclude, but are not limited to, thrombin which binds to the fXainhibitor GSK913893 (Young R., et al., Bioorg. Med. Chem. Lett. 2007,17(10): 2927-2930); plasma kallikrein which binds to the fXa inhibitorapixaban (Luettgen J., et al., Blood, 2006, 108(11) abstract 4130); andtrypsin (or its bacterial homolog subtilisin) which binds the fXainhibitor C921-78 with subnanomolar affinity (Kd=500 pM) (Betz A, etal., Biochem., 1999, 38(44):14582-14591).

In one embodiment, the derivative of the invention binds, eitherdirectly or indirectly to a factor Xa inhibitor. The terms “binding,”“binds,” “recognition,” or “recognize” as used herein are meant toinclude interactions between molecules that may be detected using, forexample, a hybridization assay. The terms are also meant to include“binding” interactions between molecules. Interactions may be, forexample, protein-protein, protein-nucleic acid, protein-small moleculeor small molecule-nucleic acid in nature. Binding may be “direct” or“indirect”. “Direct” binding comprises direct physical contact betweenmolecules. “Indirect” binding between molecules comprises the moleculeshaving direct physical contact with one or more intermediate moleculessimultaneously. For example, it is contemplated that derivatives of theinvention indirectly bind and substantially neutralize low molecularweight heparin and other indirect inhibitors of factor Xa. This bindingcan result in the formation of a “complex” comprising the interactingmolecules. A “complex” refers to the binding of two or more moleculesheld together by covalent and/or non-covalent bonds, interactions and/orforces.

“Neutralize,” “reverse,” “correct,” or “counteract” the activity of aninhibitor of fXa or similar phrases refer to inhibit or block the factorXa inhibitory or anticoagulant function of a fXa inhibitor. Such phrasesrefer to partial inhibition or blocking of the function, as well as toinhibiting or blocking most or all of fXa inhibitor activity, in vitroand/or in vivo. These terms also refer to corrections of at least about20% of fXa inhibitor dependent pharmacodynamic or surrogate markers.Examples of markers include, but are not limited to, INR, PT, aPTT, ACT,anti fXa units, thrombin generation (Technothrombin TGA,thromboelastography, CAT (calibrated automated thrombogram)) and thelike.

In certain embodiments, the factor Xa inhibitor is neutralizedsubstantially (or “corrected” as just described) meaning that itsability to inhibit factor Xa, either directly or indirectly, is reducedby at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

The term “phospholipid membrane binding” refers to an active fXa'sability to bind to the negatively charged phospholipid membrane or othercellular membrane, such as platelets, in the presence of Ca²⁺ ions. Thisbinding is mediated by the γ-carboxyglutamic acid residues in the Gladomain of fXa.

The term “reduced interaction” refers to fXa derivative's diminishedability to bind or form a complex with ions or other co-factors whichnormally binds or complexes with wild fXa. Examples of such interactioninclude but are not limited to fXa's binding with Ca²⁺ ions andphospholipid membrane, interaction with fV/fVa, or fVIII/f/VIIIa, etc.It is preferred that the interaction of a fXa derivative with the ionsor other co-factors is reduced to 50% of that of a wild fXa. Morepreferably, the interaction is reduced to 10%, 1%, and 0.1% of that of awild-type fXa. This refers to the derivatives' ability to “assemble intothe prothrombinase complex.”

“fXa inhibitor binding activity” refers to a molecule's ability to bindan inhibitor of fXa. An antidote of the present invention possesses fXainhibitor binding activity, whether it is directly or indirectly.

The term “circulating half life” or “plasma half life” refers to thetime required for the plasma concentration of an antidote thatcirculates in the plasma to reduce to half of its initial concentrationafter a single administration or following cessation of infusion.

The term “conjugated moiety” refers to a moiety that can be added to afXa derivative by forming a covalent bond with a residue of the fXaderivative. The moiety may bond directly to a residue of the fXaderivative or may form a covalent bond with a linker which in turn formsa covalent bond with a residue of the fXa derivative.

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule. Examples of such include,but are not limited to a complementarity determining region (CDR) of aheavy or light chain or a ligand binding portion thereof, a heavy chainor light chain variable region, a heavy chain or light chain constantregion, a framework (FR) region, or any portion thereof, or at least oneportion of a binding protein.

The antibodies can be polyclonal or monoclonal and can be isolated fromany suitable biological source, e.g., murine, rat, sheep and canine.

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

An effective amount” refers to the amount of derivative sufficient toinduce a desired biological and/or therapeutic result. That result canbe alleviation of the signs, symptoms, or causes of a disease, or anyother desired alteration of a biological system. In the presentinvention, the result will typically involve one or more of thefollowing: neutralization of a fXa inhibitor that has been administeredto a patient, reversal of the anticoagulant activity of the fXainhibitor, removal of the fXa inhibitor from the plasma, restoration ofhemostasis, and reduction or cessation of bleeding. The effective amountwill vary depending upon the specific antidote agent used, the specificfXa inhibitor the subject has been administered, the dosing regimen ofthe fXa inhibitor, timing of administration of the antidote, the subjectand disease condition being treated, the weight and age of the subject,the severity of the disease condition, the manner of administration andthe like, all of which can be determined readily by one of ordinaryskill in the art. One method of determining if the biological ortherapeutic result is achieved is measuring fXa inhibitor dependentpharmacodynamic or surrogate markers in a patient. The marker may be,but is not limited to, INR, PT, aPTT, ACT, anti fXa units, and thrombingeneration (Technothrombin TGA, thromboelastography, CAT (calibratedautomated thrombogram)).

The term “neutralizing amount” refers to an amount capable ofneutralizing the factor Xa inhibitor where the term “neutralizing” is asdefined herein.

As used herein, the terms “treating,” “treatment” and the like are usedherein to mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disorder or sign or symptom thereof, and/or maybe therapeutic in terms of a partial or complete cure for a disorderand/or adverse effect attributable to the disorder.

“Treating” also covers any treatment of a disorder in a mammal, andincludes: (a) preventing a disorder from occurring in a subject that maybe predisposed to a disorder, but may have not yet been diagnosed ashaving it, e.g., prevent bleeding in a patient with anticoagulantoverdose; (b) inhibiting a disorder, i.e., arresting its development, eg, inhibiting bleeding; or (c) relieving or ameliorating the disorder,e.g., reducing bleeding.

As used herein, to “treat” further includes systemic amelioration of thesymptoms associated with the pathology and/or a delay in onset ofsymptoms. Clinical and sub-clinical evidence of “treatment” will varywith the pathology, the individual and the treatment.

“Administration” can be effected in one dose, continuously orintermittently throughout the course of treatment. Methods ofdetermining the most effective means and dosage of administration areknown to those of skill in the art and will vary with the compositionused for therapy, the purpose of the therapy, the target cell beingtreated, and the subject being treated. Single or multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician. Suitable dosage formulations andmethods of administering the agents are known in the art.

The agents and compositions of the present invention can be used in themanufacture of medicaments and for the treatment of humans and otheranimals by administration in accordance with conventional procedures,such as an active ingredient in pharmaceutical compositions.

An agent of the present invention can be administered for therapy by anysuitable route, specifically by parental (including subcutaneous,intramuscular, intravenous and intradermal) administration. It will alsobe appreciated that the preferred route will vary with the condition andage of the recipient, and the disease being treated.

One can determine if the method, i.e., inhibition or reversal of afactor Xa inhibitor, is achieved by a number of in vitro assays, such asthrombin generation assay and anti-fXa units, and clinical clottingassays such as aPTT, PT and ACT.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively that are present in the natural source of themacromolecule. The term “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to polypeptides and proteins that are isolated fromother cellular proteins and is meant to encompass both purified andrecombinant polypeptides. In other embodiments, the term “isolated”means separated from constituents, cellular and otherwise, in which thecell, tissue, polynucleotide, peptide, polypeptide, protein, antibody orfragment(s) thereof, which are normally associated in nature. Forexample, an isolated cell is a cell that is separated from tissue orcells of dissimilar phenotype or genotype. As is apparent to those ofskill in the art, a non-naturally occurring polynucleotide, peptide,polypeptide, protein, antibody or fragment(s) thereof, does not require“isolation” to distinguish it from its naturally occurring counterpart.

As used herein, the term “equivalent thereof” when referring to areference protein, polypeptide or nucleic acid, intends those havingminimal homology while still maintaining desired functionality. It iscontemplated that any modified protein mentioned herein also includesequivalents thereof. For example, the homology can be, at least 75%homology and alternatively, at least 80%, or alternatively at least 85%,or alternatively at least 90%, or alternatively at least 95%, oralternatively 98% percent homology and exhibit substantially equivalentbiological activity to the reference polypeptide or protein. Apolynucleotide or polynucleotide region (or a polypeptide or polypeptideregion) has a certain percentage (for example, 80%, 85%, 90%, or 95%) of“sequence identity” to another sequence means that, when aligned, thatpercentage of bases (or amino acids) are the same in comparing the twosequences. It should be noted that when only the heavy chain of fXa (ora related serine protease) is used, the overall homology might be lowerthan 75%, such as, for example, 65% or 50% however, the desiredfunctionality remains. This alignment and the percent homology orsequence identity can be determined using software programs known in theart, for example those described in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (F. M. Ausubel et al., eds., 1987) Supplement 30, section7.7.18, Table 7.7.1. Preferably, default parameters are used foralignment. A preferred alignment program is BLAST, using defaultparameters. In particular, preferred programs are BLASTN and BLASTP,using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address:http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, vectors, isolatedDNA of any sequence, isolated RNA of any sequence, nucleic acid probesand primers. A polynucleotide can comprise modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure can be imparted before or after assembly ofthe polynucleotide. The sequence of nucleotides can be interrupted bynon-nucleotide components. A polynucleotide can be further modifiedafter polymerization, such as by conjugation with a labeling component.The term also refers to both double- and single-stranded molecules.Unless otherwise specified or required, any embodiment of this inventionthat is a polynucleotide encompasses both the double-stranded form andeach of two complementary single-stranded forms known or predicted tomake up the double-stranded form.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, or alternatively less than 25% identity, withone of the sequences of the present invention.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases (oramino acids) are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al. eds. (2007) Current Protocols in Molecular Biology.Preferably, default parameters are used for alignment. One alignmentprogram is BLAST, using default parameters. In particular, programs areBLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following Internet address:http://www.ncbi.nlm.nih.gov/blast/Blast.cgi, last accessed on Nov. 26,2007. Biologically equivalent polynucleotides are those having thespecified percent homology and encoding a polypeptide having the same orsimilar biological activity.

The term “a homolog of a nucleic acid” refers to a nucleic acid having anucleotide sequence having a certain degree of homology with thenucleotide sequence of the nucleic acid or complement thereof. A homologof a double stranded nucleic acid is intended to include nucleic acidshaving a nucleotide sequence which has a certain degree of homology withthe complement thereof. In one aspect, homologs of nucleic acids arecapable of hybridizing to the nucleic acid or complement thereof.

A “gene” refers to a polynucleotide containing at least one open readingframe (ORF) that is capable of encoding a particular polypeptide orprotein after being transcribed and translated. Any of thepolynucleotide or polypeptide sequences described herein may be used toidentify larger fragments or full-length coding sequences of the genewith which they are associated. Methods of isolating larger fragmentsequences are known to those of skill in the art.

The term “express” refers to the production of a gene product.

As used herein, “expression” refers to the process by whichpolynucleotides are transcribed into mRNA and/or the process by whichthe transcribed mRNA is subsequently being translated into peptides,polypeptides, or proteins. If the polynucleotide is derived from genomicDNA, expression may include splicing of the mRNA in an eukaryotic cell.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

A “peptide conjugate” refers to the association by covalent ornon-covalent bonding of one or more polypeptides and another chemical orbiological compound. In a non-limiting example, the “conjugation” of apolypeptide with a chemical compound results in improved stability orefficacy of the polypeptide for its intended purpose. In one embodiment,a peptide is conjugated to a carrier, wherein the carrier is a liposome,a micelle, or a pharmaceutically acceptable polymer.

“Liposomes” are microscopic vesicles consisting of concentric lipidbilayers. Structurally, liposomes range in size and shape from longtubes to spheres, with dimensions from a few hundred Angstroms tofractions of a millimeter. Vesicle-forming lipids are selected toachieve a specified degree of fluidity or rigidity of the final complexproviding the lipid composition of the outer layer. These are neutral(cholesterol) or bipolar and include phospholipids, such asphosphatidylcholine (PC), phosphatidylethanolamine (PE),phosphatidylinositol (PI), and sphingomyelin (SM) and other types ofbipolar lipids including but not limited todioleoylphosphatidylethanolamine (DOPE), with a hydrocarbon chain lengthin the range of 14-22, and saturated or with one or more double C═Cbonds. Examples of lipids capable of producing a stable liposome, alone,or in combination with other lipid components are phospholipids, such ashydrogenated soy phosphatidylcholine (HSPC), lecithin,phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin,cardiolipin, phosphatidic acid, cerebrosides,distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE) anddioleoylphosphatidylethanolamine4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal). Additionalnon-phosphorous containing lipids that can become incorporated intoliposomes include stearylamine, dodecylamine, hexadecylamine, isopropylmyristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfate, acetylpalmitate, glycerol ricinoleate, hexadecyl stearate, amphoteric acrylicpolymers, polyethyloxylated fatty acid amides, and the cationic lipidsmentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA,DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidicacid (PA), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are ableto form vesicles. Typically, liposomes can be divided into threecategories based on their overall size and the nature of the lamellarstructure. The three classifications, as developed by the New YorkAcademy Sciences Meeting, “Liposomes and Their Use in Biology andMedicine,” December 1977, are multi-lamellar vesicles (MLVs), smalluni-lamellar vesicles (SUVs) and large uni-lamellar vesicles (LUVs).

A “micelle” is an aggregate of surfactant molecules dispersed in aliquid colloid. A typical micelle in aqueous solution forms an aggregatewith the hydrophilic “head” regions in contact with surrounding solvent,sequestering the hydrophobic tail regions in the micelle center. Thistype of micelle is known as a normal phase micelle (oil-in-watermicelle). Inverse micelles have the head groups at the center with thetails extending out (water-in-oil micelle). Micelles can be used toattach a polynucleotide, polypeptide, antibody or composition describedherein to facilitate efficient delivery to the target cell or tissue.

The phrase “pharmaceutically acceptable polymer” refers to the group ofcompounds which can be conjugated to one or more polypeptides describedhere. It is contemplated that the conjugation of a polymer to thepolypeptide is capable of extending the half-life of the polypeptide invivo and in vitro. Non-limiting examples include polyethylene glycols,polyvinylpyrrolidones, polyvinylalcohols, cellulose derivatives,polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.

A “gene delivery vehicle” is defined as any molecule that can carryinserted polynucleotides into a host cell. Examples of gene deliveryvehicles are liposomes, micelles biocompatible polymers, includingnatural polymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, or viruses, such as baculovirus, adenovirus andretrovirus, bacteriophage, cosmid, plasmid, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

A polynucleotide of this invention can be delivered to a cell or tissueusing a gene delivery vehicle. “Gene delivery,” “gene transfer,”“transducing,” and the like as used herein, are terms referring to theintroduction of an exogenous polynucleotide (sometimes referred to as a“transgene”) into a host cell, irrespective of the method used for theintroduction. Such methods include a variety of well-known techniquessuch as vector-mediated gene transfer (by, e.g., viralinfection/transfection, or various other protein-based or lipid-basedgene delivery complexes) as well as techniques facilitating the deliveryof “naked” polynucleotides (such as electroporation, “gene gun” deliveryand various other techniques used for the introduction ofpolynucleotides). The introduced polynucleotide may be stably ortransiently maintained in the host cell. Stable maintenance typicallyrequires that the introduced polynucleotide either contains an origin ofreplication compatible with the host cell or integrates into a repliconof the host cell such as an extrachromosomal replicon (e.g., a plasmid)or a nuclear or mitochondrial chromosome. A number of vectors are knownto be capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors, alphavirus vectors and the like. Alphavirus vectors, such asSemliki Forest virus-based vectors and Sindbis virus-based vectors, havealso been developed for use in gene therapy and immunotherapy. See,Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 andYing, et al. (1999) Nat. Med. 5(7):823-827. In aspects where genetransfer is mediated by a retroviral vector, a vector construct refersto the polynucleotide comprising the retroviral genome or part thereof,and a therapeutic gene. As used herein, “retroviral mediated genetransfer” or “retroviral transduction” carries the same meaning andrefers to the process by which a gene or nucleic acid sequences arestably transferred into the host cell by virtue of the virus enteringthe cell and integrating its genome into the host cell genome. The viruscan enter the host cell via its normal mechanism of infection or bemodified such that it binds to a different host cell surface receptor orligand to enter the cell. As used herein, retroviral vector refers to aviral particle capable of introducing exogenous nucleic acid into a cellthrough a viral or viral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad) or adeno-associated virus (AAV), a vectorconstruct refers to the polynucleotide comprising the viral genome orpart thereof, and a transgene. Adenoviruses (Ads) are a relatively wellcharacterized, homogenous group of viruses, including over 50 serotypes.See, e.g., International PCT Application No. WO 95/27071. Ads do notrequire integration into the host cell genome. Recombinant Ad derivedvectors, particularly those that reduce the potential for recombinationand generation of wild-type virus, have also been constructed. See,International PCT Application Nos. WO 95/00655 and WO 95/11984.Wild-type AAV has high infectivity and specificity integrating into thehost cell's genome. See, Hermonat and Muzyczka (1984) Proc. Natl. Acad.Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol.8:3988-3996.

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are well known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include DNA/liposome complexes, micelles andtargeted viral protein-DNA complexes. Liposomes that also comprise atargeting antibody or fragment thereof can be used in the methods ofthis invention. To enhance delivery to a cell, the nucleic acid orproteins of this invention can be conjugated to antibodies or bindingfragments thereof which bind cell surface antigens, e.g., a cell surfacemarker found on stem cells or cardiomyocytes. In addition to thedelivery of polynucleotides to a cell or cell population, directintroduction of the proteins described herein to the cell or cellpopulation can be done by the non-limiting technique of proteintransfection, alternatively culturing conditions that can enhance theexpression and/or promote the activity of the proteins of this inventionare other non-limiting techniques.

The phrase “solid support” refers to non-aqueous surfaces such as“culture plates” “gene chips” or “microarrays.” Such gene chips ormicroarrays can be used for diagnostic and therapeutic purposes by anumber of techniques known to one of skill in the art. In one technique,oligonucleotides are arrayed on a gene chip for determining the DNAsequence by the hybridization approach, such as that outlined in U.S.Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of this inventioncan be modified to probes, which in turn can be used for detection of agenetic sequence. Such techniques have been described, for example, inU.S. Pat. Nos. 5,968,740 and 5,858,659. A probe also can be affixed toan electrode surface for the electrochemical detection of nucleic acidsequences such as described by Kayem et al. U.S. Pat. No. 5,952,172 andby Kelley et al. (1999) Nucleic Acids Res. 27:4830-4837.

Various “gene chips” or “microarrays” and similar technologies are knowin the art. Examples of such include, but are not limited to, LabCard(ACLARA Bio Sciences Inc.); GeneChip (Affymetric, Inc); LabChip (CaliperTechnologies Corp); a low-density array with electrochemical sensing(Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); OmniGrid (Gene Machines); Q Array (Genetix Ltd.); a high-throughput,automated mass spectrometry systems with liquid-phase expressiontechnology (Gene Trace Systems, Inc.); a thermal jet spotting system(Hewlett Packard Company); Hyseq HyChip (Hyseq, Inc.); BeadArray(Illumina, Inc.); GEM (Incyte Microarray Systems); a high-throughputmicroarrying system that can dispense from 12 to 64 spots onto multipleglass slides (Intelligent Bio-Instruments); Molecular BiologyWorkstation and NanoChip (Nanogen, Inc.); a microfluidic glass chip(Orchid biosciences, Inc.); BioChip Arrayer with four PiezoTippiezoelectric drop-on-demand tips (Packard Instruments, Inc.); FlexJet(Rosetta Inpharmatic, Inc.); MALDI-TOF mass spectrometer (Sequnome);ChipMaker 2 and ChipMaker 3 (TeleChem International, Inc.); andGenoSensor (Vysis, Inc.) as identified and described in Heller (2002)Annu Rev. Biomed. Eng. 4:129-153. Examples of “gene chips” or a“microarrays” are also described in U.S. Patent Publ. Nos.:2007-0111322, 2007-0099198, 2007-0084997, 2007-0059769 and 2007-0059765and U.S. Pat. Nos. 7,138,506, 7,070,740, and 6,989,267.

In one aspect, “gene chips” or “microarrays” containing probes orprimers homologous to a polynucleotide, polypeptide or antibodydescribed herein are prepared. A suitable sample is obtained from thepatient, extraction of genomic DNA, RNA, protein or any combinationthereof is conducted and amplified if necessary. The sample is contactedto the gene chip or microarray panel under conditions suitable forhybridization of the gene(s) or gene product(s) of interest to theprobe(s) or primer(s) contained on the gene chip or microarray. Theprobes or primers may be detectably labeled thereby identifying thegene(s) of interest. Alternatively, a chemical or biological reactionmay be used to identify the probes or primers which hybridized with theDNA or RNA of the gene(s) of interest. The genotypes or phenotype of thepatient is then determined with the aid of the aforementioned apparatusand methods.

Other non-limiting examples of a solid phase support include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble. The support material may have virtually anypossible structural configuration so long as the coupled molecule iscapable of binding to a polynucleotide, polypeptide or antibody. Thus,the support configuration may be spherical, as in a bead, orcylindrical, as in the inside surface of a test tube, or the externalsurface of a rod. Alternatively, the surface may be flat such as asheet, test strip, etc. or alternatively polystyrene beads. Thoseskilled in the art will know many other suitable carriers for bindingantibody or antigen, or will be able to ascertain the same by use ofroutine experimentation.

“Eukaryotic cells” comprise all of the life kingdoms except monera. Theycan be easily distinguished through a membrane-bound nucleus. Animals,plants, fungi, and protists are eukaryotes or organisms whose cells areorganized into complex structures by internal membranes and acytoskeleton. The most characteristic membrane-bound structure is thenucleus. A eukaryotic host, including, for example, yeast, higher plant,insect and mammalian cells, or alternatively from a prokaryotic cells asdescribed above. Non-limiting examples include simian, bovine, porcine,murine, rats, avian, reptilian and human.

“Prokaryotic cells” that usually lack a nucleus or any othermembrane-bound organelles and are divided into two domains, bacteria andarchaea. Additionally, instead of having chromosomal DNA, these cells'genetic information is in a circular loop called a plasmid. Bacterialcells are very small, roughly the size of an animal mitochondrion (about1-2 μm in diameter and 10 μm long). Prokaryotic cells feature threemajor shapes: rod shaped, spherical, and spiral. Instead of goingthrough elaborate replication processes like eukaryotes, bacterial cellsdivide by binary fission. Examples include but are not limited tobacillus bacteria, E. coli bacterium, and Salmonella bacterium.

The term “human antibody” as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo).However, the term “human antibody” as used herein, is not intended toinclude antibodies in which CDR sequences derived from the germline ofanother mammalian species, such as a mouse, have been grafted onto humanframework sequences. Thus, as used herein, the term “human antibody”refers to an antibody in which substantially every part of the protein(e.g., CDR, framework, C_(L), C_(H) domains (e.g., C_(H1), C_(H2),C_(H3)), hinge, (VL, VH)) is substantially non-immunogenic in humans,with only minor sequence changes or variations. Similarly, antibodiesdesignated primate (monkey, baboon, chimpanzee, etc.), rodent (mouse,rat, rabbit, guinea pig, hamster, and the like) and other mammalsdesignate such species, sub-genus, genus, sub-family, family specificantibodies. Further, chimeric antibodies include any combination of theabove. Such changes or variations optionally and preferably retain orreduce the immunogenicity in humans or other species relative tonon-modified antibodies. Thus, a human antibody is distinct from achimeric or humanized antibody. It is pointed out that a human antibodycan be produced by a non-human animal or prokaryotic or eukaryotic cellthat is capable of expressing functionally rearranged humanimmunoglobulin (e.g., heavy chain and/or light chain) genes. Further,when a human antibody is a single chain antibody, it can comprise alinker peptide that is not found in native human antibodies. Forexample, an Fv can comprise a linker peptide, such as two to about eightglycine or other amino acid residues, which connects the variable regionof the heavy chain and the variable region of the light chain. Suchlinker peptides are considered to be of human origin.

As used herein, a human antibody is “derived from” a particular germlinesequence if the antibody is obtained from a system using humanimmunoglobulin sequences, e.g., by immunizing a transgenic mousecarrying human immunoglobulin genes or by screening a humanimmunoglobulin gene library. A human antibody that is “derived from” ahuman germline immunoglobulin sequence can be identified as such bycomparing the amino acid sequence of the human antibody to the aminoacid sequence of human germline immunoglobulins. A selected humanantibody typically is at least 90% identical in amino acids sequence toan amino acid sequence encoded by a human germline immunoglobulin geneand contains amino acid residues that identify the human antibody asbeing human when compared to the germline immunoglobulin amino acidsequences of other species (e.g., murine germline sequences). In certaincases, a human antibody may be at least 95%, or even at least 96%, 97%,98%, or 99% identical in amino acid sequence to the amino acid sequenceencoded by the germline immunoglobulin gene. Typically, a human antibodyderived from a particular human germline sequence will display no morethan 10 amino acid differences from the amino acid sequence encoded bythe human germline immunoglobulin gene. In certain cases, the humanantibody may display no more than 5, or even no more than 4, 3, 2, or 1amino acid difference from the amino acid sequence encoded by thegermline immunoglobulin gene.

A “human monoclonal antibody” refers to antibodies displaying a singlebinding specificity which have variable and constant regions derivedfrom human germline immunoglobulin sequences. The term also intendsrecombinant human antibodies. Methods to making these antibodies aredescribed herein.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the antibody, e.g., from a transfectoma,antibodies isolated from a recombinant, combinatorial human antibodylibrary, and antibodies prepared, expressed, created or isolated by anyother means that involve splicing of human immunoglobulin gene sequencesto other DNA sequences. Such recombinant human antibodies have variableand constant regions derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo. Methods to makingthese antibodies are described herein.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG1) that is encoded by heavy chain constant region genes.

The terms “polyclonal antibody” or “polyclonal antibody composition” asused herein refer to a preparation of antibodies that are derived fromdifferent B-cell lines. They are a mixture of immunoglobulin moleculessecreted against a specific antigen, each recognizing a differentepitope.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

As used herein, the term “label” intends a directly or indirectlydetectable compound or composition that is conjugated directly orindirectly to the composition to be detected, e.g., polynucleotide orprotein such as an antibody so as to generate a “labeled” composition.The term also includes sequences conjugated to the polynucleotide thatwill provide a signal upon expression of the inserted sequences, such asgreen fluorescent protein (GFP) and the like. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable. The labelscan be suitable for small scale detection or more suitable forhigh-throughput screening. As such, suitable labels include, but are notlimited to radioisotopes, fluorochromes, chemiluminescent compounds,dyes, and proteins, including enzymes. The label may be simply detectedor it may be quantified. A response that is simply detected generallycomprises a response whose existence merely is confirmed, whereas aresponse that is quantified generally comprises a response having aquantifiable (e.g., numerically reportable) value such as an intensity,polarization, and/or other property. In luminescence or fluorescenceassays, the detectable response may be generated directly using aluminophore or fluorophore associated with an assay component actuallyinvolved in binding, or indirectly using a luminophore or fluorophoreassociated with another (e.g., reporter or indicator) component.

Examples of luminescent labels that produce signals include, but are notlimited to bioluminescence and chemiluminescence. Detectableluminescence response generally comprises a change in, or an occurrenceof, a luminescence signal. Suitable methods and luminophores forluminescently labeling assay components are known in the art anddescribed for example in Haugland, Richard P. (1996) Handbook ofFluorescent Probes and Research Chemicals (6^(th) ed.). Examples ofluminescent probes include, but are not limited to, aequorin andluciferases.

Examples of suitable fluorescent labels include, but are not limited to,fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin,coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, LuciferYellow, Cascade Blue™, and Texas Red. Other suitable optical dyes aredescribed in the Haugland, Richard P. (1996) Handbook of FluorescentProbes and Research Chemicals (6^(th) ed.).

In another aspect, the fluorescent label is functionalized to facilitatecovalent attachment to a cellular component present in or on the surfaceof the cell or tissue such as a cell surface marker. Suitable functionalgroups, including, but not are limited to, isothiocyanate groups, aminogroups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonylhalides, all of which may be used to attach the fluorescent label to asecond molecule. The choice of the functional group of the fluorescentlabel will depend on the site of attachment to either a linker, theagent, the marker, or the second labeling agent.

III. Unit Dose Formulations and Methods of their Use

One aspect of the invention provides a unit dose formulation comprisinga pharmaceutically acceptable carrier and a two chain polypeptidecomprising the amino acid sequence of SEQ ID NO. 13 (r-Antidote) or apolypeptide having at least 80% homology to SEQ ID NO. 13, in an amountfrom about 10 milligrams to about 2 grams. The invention is based on thesurprising discovery that the r-Antidote is capable of neutralizing avariety of factor Xa inhibitors, such as betrixaban, rivaroxaban, lowmolecular weight heparin, enoxaparin, and apixaban at a certain dose inrats and monkeys. This data was then extrapolated using modeling toarrive at the dose for humans capable of neutralizing the inhibitor asexplained in Examples 25 and 26 below.

In certain aspects, the formulation is administered in an amount of fromabout 10 milligrams (mg) to about 2 grams (g). Other amountscontemplated by this invention include from about 100 mg to about 1.5 g;from about 200 mg to about 1 g; and from about 400 mg to about 900 mg.

In another embodiment, the unit dose formulation is administered in aneutralizing amount that is at least about a 1:1 fold molar ratio ofcirculating concentration of polypeptide over circulating concentrationof the factor Xa inhibitor for a period of at least about 30 minutes. Inother embodiments the molar ratio is about 1:1 or about 2:1 or about4:1.

The formulation when administered neutralizes the factor Xa inhibitor byat least about 20%, or by at least about 50%, or by at least about 75%,or by at least about 90%, or by at least about 95%.

“Pharmaceutically acceptable carriers” refers to any diluents,excipients, or carriers that may be used in the compositions of theinvention. Pharmaceutically acceptable carriers include saline, ionexchangers, alumina, aluminum stearate, lecithin, serum proteins, suchas human serum albumin, buffer substances, such as phosphates, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field. They are preferably selected with respect to theintended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

In one embodiment, the formulation comprises saline and the antidote ispresent in a concentration of from about 0.2 to about 10 mg ofpolypeptide per milliliter of saline. In another embodiment, theconcentration is from about 2 to about 6 mg per milliliter of saline. Instill another embodiment, the concentration is about 2 mg per milliliterof saline.

The formulations of the invention can be manufactured by methods wellknown in the art such as conventional granulating, mixing, dissolving,encapsulating, lyophilizing, or emulsifying processes, among others.Compositions may be produced in various forms, including granules,precipitates, or particulates, powders, including freeze dried, rotarydried or spray dried powders, amorphous powders, injections, emulsions,elixirs, suspensions or solutions. Formulations may optionally containstabilizers, pH modifiers, surfactants, bioavailability modifiers andcombinations of these.

In one embodiment, the antidote is lyophilized. Methods for lyophilizingpolypeptides is well known in the art.

Pharmaceutical formulations may also be prepared as liquid suspensionsor solutions using a sterile liquid, such as oil, water, alcohol, andcombinations thereof. Pharmaceutically suitable surfactants, suspendingagents or emulsifying agents, may be added for oral or parenteraladministration. Suspensions may include oils, such as peanut oil, sesameoil, cottonseed oil, corn oil and olive oil. Suspension preparation mayalso contain esters of fatty acids, such as ethyl oleate, isopropylmyristate, fatty acid glycerides and acetylated fatty acid glycerides.Suspension formulations may include alcohols, such as ethanol, isopropylalcohol, hexadecyl alcohol, glycerol and propylene glycol. Ethers, suchas poly(ethyleneglycol), petroleum hydrocarbons, such as mineral oil andpetrolatum, and water may also be used in suspension formulations.

One formulation contemplated by use of the invention is predicated uponthe formulation for a commercially available recombinant humancoagulation Factor VIIa (rFVIIa). This formulation employs a lyophilizedpolypeptide and contains the following additional ingredients:

Contents 1.2 mg vial 4.8 mg vial Polypeptide (antidote) 1200 micrograms4800 micrograms sodium chloride 6 mg 23 mg calcium chloride 3 mg 12 mgdihydrate glycylglycine 3 mg 11 mg polysorbate 80 0.2 mg 0.6 mg mannitol60 mg 240 mg

In one aspect, the invention is directed to methods of selectivelybinding and inhibiting an exogenously administered factor Xa inhibitorin a subject undergoing anticoagulant therapy with a factor Xa inhibitorcomprising administering to the subject a unit dose formulation of theinvention.

In another aspect, the invention is directed to a method of preventing,reducing, or ceasing bleeding in a subject undergoing anticoagulanttherapy with a factor Xa inhibitor comprising administering to thesubject a unit dose formulation of the invention.

In still another aspect, the invention is directed to a method forcorrecting fXa inhibitor dependent pharmacodynamic or surrogate markersin a patient undergoing anticoagulant therapy with a factor Xa inhibitorcomprising administering to the subject a unit dose formulation of theinvention. The pharmacodynamic or surrogate marker may be selected fromthe consisting of INR, PT, aPTT, ACT, anti fXa units, and thrombingeneration (Technothrombin TGA, thromboelastography, CAT (calibratedautomated thrombogram)).

The formulations are for administration to a mammal, preferably a humanbeing. Such formulations of the invention may be administered in avariety of ways, preferably parenterally.

It is contemplated that in order to quickly reverse the anticoagulantactivity of a fXa inhibitor present in a patient's plasma in a emergencysituation, the antidote of this invention can or may be administered tothe systemic circulation via parental administration. The term“parenteral” as used herein includes subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional and intracranial injection orinfusion techniques. However, in cases where the fXa inhibitor beingneutralized has a long plasma half life, a continuous infusion or asustained release formulation may be required to bind to the fXainhibitor and such free up the active fXa prior to the clearance of thefXa inhibitor from the body. Therefore, in one aspect, the formulationis administered to the subject as a bolus. In another aspect, theformulation is administered by infusion. In another aspect, theformulation is administered by a combination of bolus and infusion.

The formulation is administered until bleeding has substantially ceased.It is contemplated that the infusion can be administered for about 6hours, or about 6 to about 12 hours, or about 12 to about 24 hours or 48hours.

In some embodiments, about 10% to about 20% of the total dose and theremainder would be infused over the time periods just mentioned.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation. Compounds may be formulated for parenteraladministration by injection such as by bolus injection or continuousinfusion. A unit dosage form for injection may be in ampoules or inmulti-dose containers.

In addition to dosage forms described above, pharmaceutically acceptableexcipients and carriers and dosage forms are generally known to thoseskilled in the art and are included in the invention. It should beunderstood that a specific dosage and treatment regimen for anyparticular patient will depend upon a variety of factors, including theactivity of the specific antidote employed, the age, body weight,general health, sex and diet, renal and hepatic function of the patient,and the time of administration, rate of excretion, drug combination,judgment of the treating physician or veterinarian and severity of theparticular disease being treated.

IV. Antidotes

Additional antidotes contemplated for use in the formulation and methodsof the invention are found below.

Factor Xa Derivatives

One aspect of the present invention is the use of fXa derivatives, suchas Gla-domain deficient fXa or des-Gla fXa, as safe and effectiveantidotes to substantially neutralize the activity of an inhibitor ofthe coagulation fXa to prevent or stop bleeding. It is contemplated thatthe antidotes of the present invention will be useful in reversing theanticoagulant effect of a fXa inhibitor, especially an activesite-directed small molecule inhibitor.

It is contemplated that an antidote to a fXa inhibitor has reduced or noprocoagulant activity but is capable of binding with a fXa inhibitor. Itis contemplated that such limited activity permits dosing of theantidote at a level greater than the circulating wild-type fXa. CertainfXa derivatives, such as des-Gla fXa and Gla-deficient fXa, are suitableantidotes of this invention. Besides having reduced or diminishedprocoagulant activity, antidotes of the present invention should also besubstantially non-immunogenic to the subject. An antidote may contain acombination of two or more of the above mutations and/or modifications.In addition, any of the above fXa derivatives may be administered aloneor in combination with one another.

Factor Xa is a serine protease in the blood coagulation pathwayresponsible for converting prothrombin to thrombin. It is produced fromthe inactive factor X upon activation by either the intrinsic Xase(complex formed by factor IXa with its cofactor, factor VIIIa) or theextrinsic Xase (complex formed by factor VIIa with its cofactor, tissuefactor). Activated fX (fXa) may undergo further autocatalytic cleavageat the C-terminal of its heavy chain, converting fXaα to the subformfXaβ (Jesty, J et al. J. Biol. Chem. 1975, 250(12):4497-4504). Both fXaαand fXaβ are suitable materials for the present invention. fXa itselfconverts prothrombin at a slow rate that is not sufficient forsupporting coagulation. Only when it forms a prothrombinase complex withcofactors Ca²⁺, phospholipid, and factor Va, fXa can activateprothrombin at a rate rapid enough to support coagulation (Skogen, W.F., et al., J. Biol. Chem. 1984, 259(4):2306-10). The complex requiresbinding between the negatively charged phospholipid andγ-carboxyglutamic acid residues in the Gla domain of fXa via Ca²⁺bridging.

Therefore, although the Gla domain does not contain the active site offXa, it enables fXa to form the prothrombinase complex through theγ-carboxyglutamic acid residues. This is demonstrated by selectiveremoval of fXa Gla-domain by chymotrypsin digestion (see FIG. 7 andExample 1). Clotting assays were performed on fXa during the time courseof cleavage of the Gla domain by chymotrypsin digestion. It has beenreported (Skogen et al. J. Biol. Chem. 1984, 259(4):2306-10) that areconstituted prothrombinase complex comprising of Gla-domainless fXa,fVa, phospholipids and calcium ions produces thrombin at a significantlyreduced rate (0.5% product generated compared to control complexcontaining native fXa). As shown in FIG. 7, fXa's activity in clotformation was partially reduced after the fXa was digested bychymotrypsin for 15 minutes and the activity was completely lost after30 minutes of digestion. Undercarboxylated or decarboxylated fXa, whichlack the appropriate gamma-carboxyglutamic acid residues required forcalcium ion dependent membrane binding, have thus been found to beincapable of membrane dependent coagulation complex assembly and notsupport blood clotting (Mann, K G et al., Blood, 1990, 76: 1-16).

It has also been established that Gla-domain deficient fXa is capable ofbinding active site-directed inhibitors of fXa. (Brandstetter, H et al,J. Bio. Chem., 1996, 271:29988-29992). There have been reports ofcrystallography of small molecule fXa inhibitor bound to des-Gla humanfXa, which have provided structural description of the active site cleft(Brandstetter, J. Bio. Chem., 1996, 271:29988-29992 and Roehrig, J. Med.Chem. 2005, 48(19):5900-8). FIG. 8 shows that a des-Gla anhydro-fXaexhibited a binding affinity of 0.319 nM with a fXa inhibitorbetrixaban, comparable to that of native fXa.

It has now been discovered that des-Gla fXa, and other fXa derivativesthat have reduced procoagulant activity but are capable of fXa inhibitorbinding, can be used as an antidote to a fXa inhibitor. As shown in FIG.9, the des-Gla anhydro-fXa exhibited complete reversion of betrixaban'santicoagulant activity at a concentration of 680 nM. As detailed inExample 2, the thrombin generation was initiated by adding TF-containingreagent (Innovin) and, thus, indicative of coagulation factors functionin the extrinsic coagulation pathway. It has also been demonstrated inExamples 9-13, that the recombinant antidote is useful to reverse a widevariety of anticoagulants.

Clotting prolongation assays with the activated partial thromboplastintime (aPTT) reagent (Actin FS) that determine the function of thecoagulation factor in the intrinsic coagulation pathway also indicatethat the des-Gla anhydro-fXa possess antidote activity. FIG. 10 showsthe dose responsive antidote effect of des-Gla anhydro-fXa against 250nM of betrixaban, with complete reversion at 600 nM. FIG. 11 shows thatdes-Gla anhydro-fXa was also capable of reversing the anticoagulantactivity of another fXa inhibitor, enoxaparin. FIG. 12 shows thatdes-Gla anhydro-fXa did not exhibit significant antidote activityagainst a direct thrombin inhibitor argatroban. Thus, the des-Glaanhydro-fXa is a selective antidote for fXa inhibitors and is capable ofrestoring fXa procoagulant activity initiated either by the extrinsic orthe intrinsic pathway.

Further, the antidote activity of des-Gla anhydro-fXa was demonstratedby the aPTT prolongation assays measured with a traditional coagulationtimer. As shown in FIG. 13, des-Gla anhydro-fXa itself has no effect onaPTT of control plasma at the highest concentrations tested (2576 nM).400 nM of betrixaban extended aPTT more than two folds. Thisanticoagulant effect of betrixaban is reversed by des-Gla anhydro-fXa ina dose-responsive manner, with return of aPTT to near normal level ofcontrol plasma at antidote concentrations higher than 1610 nM.

It is contemplated that further truncations at the fXa light chain, forexample, additional deletion of the EGF1 domain, EGF1 plus EGF2 domains,or fragments thereof, and inactive fXa with only the heavy chain may beuseful antidotes of this invention.

Gla-domain deficient fXa does not support normal coagulation underphysiologically relevant concentration. However, the protein has theability of cleaving many substrates and causing clotting at higherconcentrations. For example, Skogen et al. (Skogen, W. F., et al., J.Biol. Chem. 1984, 259(4):2306-10) showed that bovine des-Gla fXa hasabout 0.5-1.0% prothrombinase complex activity relative to the wild typefXa. Thus, modifications that further reduce or completely eliminate afXa derivative's procoagulant activity is contemplated by methods of theinvention. Such modification may be, for example, in a fXa's catalyticdomain.

Several ways of modifying the catalytic domain in the fXa heavy chain toreduce its procoagulant activity are contemplated. The active siteresidue S379 of fXa (as shown in SEQ ID No. 7), for example, can beselectively replaced by dehydro-alanine (see Example 1) or alanine (seeExample 6) to reduce or eliminate the procoagulant activity. It is alsoknown that complex formation between fXa and a reagent targeting fXa'sexosite may block the macromolecular binding ability of fXa, thusreducing its procoagulant activity while retaining small moleculebinding ability in the active site. This exosite targeting reagentincludes, without limitation, monoclonal antibodies targeting a regionremoved from the active site (Wilkens, M and Krishnaswamy, S, J. Bio.Chem., 2002, 277 (11), 9366-9374), or α-2-macroglobulin. It has beenknown that the α-2-macroglobulin-serine protease complex, such as withtrypsin, thrombin or fXa, is capable of binding small moleculesubstrates (Kurolwa, K. et al., Clin. Chem. 1989, 35(11), 2169-2172).

It is also known that an inactive fXa with modifications solely in theheavy chain while keeping its light chain unchanged would act as aninhibitor of prothrombinase (Hollenbach, S. et al., Thromb. Haemost.,1994, 71(3), 357-62) because it interferes with procoagulant activity ofnormal fXa as shown in FIG. 6. Therefore, in one embodiment, the fXaderivative has modifications both in the light chain and heavy chain. Ithas been discovered that these modifications reduce or eliminate bothprocoagulant and anticoagulant activities while retaining the inhibitorsbinding ability of the fXa derivative.

Several methods can be used to produce Gla-domain deficient fXaderivatives or other fXa derivatives described herein. For example, theGla-domain may be completely removed via chymotryptic cleavage,producing Gla-domainless fXa. Alternatively, a Gla-domainless fX may beproduced by chymotryptic cleavage of native fX. The Gla-domainless fXmay then be converted to Gla-domainless fXa by a fX activator. fX may beisolated from plasma of the same or a different species as the subjectto be treated. Bovine fX, for example, has been shown to be functionalin human plasma assays. Examples of a fX activator include, withoutlimitation, a snake venom, such as Russell's viper venom, and complexesof fVIIa/tissue factor or fIXa/fVIIIa. Such means is known to a personof skill in the art. For example, Rudolph A. E. et al. has reported arecombinant fXa produced from a recombinant factor X (fX) with a singlesubstitution of Arg347 by Glutamine (fXR347N) (Biochem. 2000, 39 (11):2861-2867). In one embodiment, the fXa derivatives produced fromnon-human sources are non-immunogenic or substantially non-immunogenic.Example 7 also provides a method of producing a recombinant antidotehaving the amino acid sequence of SEQ ID NO. 12.

The fXa derivatives may also be purified from human plasma, or may beproduced by recombinant DNA method where an appropriate gene for the fXaderivative is expressed in a suitable host organism. Expression andpurification of recombinant fXa has been reported by several groups,see, e.g., Larson, P. J., et al., Biochem., 1998, 37:5029-5038, andCamire, R. M., et al., Biochem., 2000, 39, 14322-14329 for producingrecombinant fX; Wolf, D. L. et al., J. Bio. Chem., 1991,266(21):13726-13730 for producing recombinant fXa. Modified fXa may beprepared according to these procedures using a genetically modified cDNAhaving a nucleotide sequence encoding the desired fXa mutant. Example 6gives more details for direct expression of a Gla-domainless fXa-5379mutant with functional activity as an antidote.

It is contemplated that active-site mutated or modified fXa withdeficient Gla-domain, such as under-carboxylated fXa, may also be usefulas fXa inhibitor antidote. Under-carboxylated fXa may be prepared byrecombinant means by withholding vitamin K derivatives during proteinexpression (vitamin K derivatives are needed for post translationalmodification to form the Gla residues) or by adding vitamin Kantagonists such as warfarin during tissue culture. Decarboxylated fXacan be prepared by heating (Bajaj P., J. Biol. Chem., 1982,257(7):3726-3731) or by proteolytic digestion by chymotrypsin (MoritaT., et al., J. Biol. Chem., 1986, 261(9):4015-4023). The antidote mayalso be generated in prokaryotic systems followed by in vitro refoldingor constitution of the fXa inhibitor binding site.

The Gla residues can also be chemically modified to remove the carboxylgroup responsible for calcium ion dependent membrane binding. Forexample, the carboxyl groups on the Gla residues may be selectivelyremoved under decarboxylation conditions or may be capped, for example,by esterification or amination. It is desirable that such esterificationor amination be resistant to in vivo hydrolysis so that the modified fXais not readily converted to active fXa, which may cause thrombosis.

Other mutants or derivatives of fXa may also be useful antidotes of thisinvention. In one embodiment, this invention encompasses use of mutantsdescribed in Peter J. Larson et al., Biochem., 1998, 37:5029-5038 as fXainhibitor antidotes.

In another embodiment, this invention encompasses use of catalyticallyinactive fXa mutants to prepare fXa inhibitor antidotes. For example,mutants described in Sinha, U., et al., Protein Expression and Purif.,1992, 3:518-524 rXai, mutants with chemical modifications, such asdehydro-alanine (anhydro fXa), as described in Nogami, et al., J. Biol.Chem. 1999, 274(43):31000-7. FXa with active site serine (Ser379 in fXnumbering as shown in SEQ ID NO. 7, and Ser195 in chymotrypsinnumbering) replaced with alanine (fXa-S379A in fX numbering, orfXa-S195A in chymotrypsin numbering), where the procoagulant activitywas eliminated, may also be used as fXa inhibitor antidotes. Theinvention also envisions fXa derivatives with the active site serineresidue irreversibly acylated which is still capable of binding smallmolecule inhibitors. FXa with the active site serine reversibly acylatedhas been reported by Wolf, et al., Blood, 1995, 86(11):4153-7. Suchreversible acylation, however, is capable of time dependent productionof active fXa and may lead to an excess of active fXa over a timeperiod. The deacylation rate may be reduced by strategies similar tothose described in Lin P. H. et al., Thrombosis Res., 1997, 88(4),365-372. For example, fXa molecules with Ser379 (Ser195 in chymotrypsinnumbering) acylated by 4-methoxybenzyl and 3-bromo-4-methoxybenzylgroups recover less than 50% of their original activity when incubatedin a buffer having pH 7.5 at 37° C. for 4 hours.

One embodiment is directed to the use of fXa derivatives with mutationsat fXa residues known to be important for fXa interaction with cofactorfV/fVa. Such residues include, without limitation, Arg306, Glu310,Arg347, Lys351, or Lys414 (SEQ ID NOS. 3 and 7, these amino acidscorrespond to Arg125, Glu129, Arg165, Lys169, Lys230 in the chymotrypsinnumbering). Examples of such mutants are reported in Rudolph, A. E. etal., J. Bio. Chem., 2001, 276:5123-5128. In addition, mutations at fXaresidues known to be important for fVIII/fVIIIa interaction, such asArg424 in SEQ ID NOS. 3 and 7 (Arg240 in chymotrypsin numbering), mayalso be used as fXa inhibitor antidotes. Examples of such mutants aredescribed in Nogami, K. et al., J. Biol. Chem., 2004,279(32):33104-33113.

Other modification of active site residues of fXa or residues known tobe important for serine protease interactions may also lead to usefulantidotes of this invention, for example, replacement of Glu216, Glu218,and Arg332 in SEQ ID NOS. 3 and 7 (Glu37, Glu39, and Arg150 inchymotrypsin numbering, respectively) with other amino acid residues.

In one embodiment, the residual procoagulant activity of an antidote, asassessed by amidolytic substrate cleavage assay, be <1%, preferably<0.1%, more preferably <0.05% of human plasma derived native fXa. Forexample, there is no measurable procoagulant activity for recombinantfXa-S379A when the active site Ser379 (S195 in chymotrypsin numbering)is replaced by an alanine residue as measured by clotting assays.

The invention further relates to nucleic acid sequences, in particularDNA sequences, which code for the fXa derivatives described above. Thesecan easily be determined by translating the polypeptide sequence backinto the corresponding DNA sequence in accordance with the genetic code.Codons preferably used are those which lead to good expression in therequired host organism. The nucleic acid sequences can be preparedeither by site-specific mutagenesis starting from the natural fXa genesequence or else by complete DNA synthesis.

Polypeptides of the Invention

In certain aspects, the invention is related to an isolated polypeptidecomprising the amino acid sequence of SEQ ID NO. 13 or 15. Alsoencompassed by this invention are polypeptides having at least 80%homology to SEQ ID NO. 13 or 15.

Polypeptides comprising the amino acid sequences of the invention can beprepared by expressing polynucleotides encoding the polypeptidesequences of this invention in an appropriate host cell. This can beaccomplished by methods of recombinant DNA technology known to thoseskilled in the art. Accordingly, this invention also provides methodsfor recombinantly producing the polypeptides of this invention in aeukaryotic or prokaryotic host cells. The proteins and polypeptides ofthis invention also can be obtained by chemical synthesis using acommercially available automated peptide synthesizer such as thosemanufactured by Perkin Elmer/Applied Biosystems, Inc., Model 430A or431A, Foster City, Calif., USA. The synthesized protein or polypeptidecan be precipitated and further purified, for example by highperformance liquid chromatography (HPLC). Accordingly, this inventionalso provides a process for chemically synthesizing the proteins of thisinvention by providing the sequence of the protein and reagents, such asamino acids and enzymes and linking together the amino acids in theproper orientation and linear sequence.

It is known to those skilled in the art that modifications can be madeto any peptide to provide it with altered properties. Polypeptides ofthe invention can be modified to include unnatural amino acids. Thus,the peptides may comprise D-amino acids, a combination of D- and L-aminoacids, and various “designer” amino acids (e.g., β-methyl amino acids,C-α-methyl amino acids, and N-α-methyl amino acids, etc.) to conveyspecial properties to peptides. Additionally, by assigning specificamino acids at specific coupling steps, peptides with α-helices, βturns, β sheets, α-turns, and cyclic peptides can be generated.Generally, it is believed that α-helical secondary structure or randomsecondary structure is preferred.

In a further embodiment, subunits of polypeptides that confer usefulchemical and structural properties will be chosen. For example, peptidescomprising D-amino acids may be resistant to L-amino acid-specificproteases in vivo. Modified compounds with D-amino acids may besynthesized with the amino acids aligned in reverse order to produce thepeptides of the invention as retro-inverso peptides. In addition, thepresent invention envisions preparing peptides that have better definedstructural properties, and the use of peptidomimetics, andpeptidomimetic bonds, such as ester bonds, to prepare peptides withnovel properties. In another embodiment, a peptide may be generated thatincorporates a reduced peptide bond, i.e., R₁—CH₂NH—R₂, where R₁, and R₂are amino acid residues or sequences. A reduced peptide bond may beintroduced as a dipeptide subunit. Such a molecule would be resistant topeptide bond hydrolysis, e.g., protease activity. Such molecules wouldprovide ligands with unique function and activity, such as extendedhalf-lives in vivo due to resistance to metabolic breakdown, or proteaseactivity. Furthermore, it is well known that in certain systemsconstrained peptides show enhanced functional activity (Hruby (1982)Life Sciences 31:189-199 and Hruby et al. (1990) Biochem J.268:249-262); the present invention provides a method to produce aconstrained peptide that incorporates random sequences at all otherpositions.

The following non-classical amino acids may be incorporated in thepeptides of the invention in order to introduce particularconformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate(Kazrnierski et al. (1991) J. Am. Chem. Soc. 113:2275-2283);(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine(Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41):5769-5772);2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D.Thesis, University of Arizona);hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al.(1989) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylicacid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); andHIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep.Protein Res. 42(1):68-77) and (Dharanipragada et al. (1992) Acta.Crystallogr. C. 48:1239-1241).

The following amino acid analogs and peptidomimetics may be incorporatedinto a peptide to induce or favor specific secondary structures: LL-Acp(LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducingdipeptide analog (Kemp et al. (1985) J. Org. Chem. 50:5834-5838);β-sheet inducing analogs (Kemp et al. (1988) Tetrahedron Lett.29:5081-5082); β-turn inducing analogs (Kemp et al. (1988) TetrahedronLett. 29:5057-5060); α-helix inducing analogs (Kemp et al. (1988)Tetrahedron Lett. 29:4935-4938); α-turn inducing analogs (Kemp et al.(1989) J. Org. Chem. 54:109:115); analogs provided by the followingreferences: Nagai and Sato (1985) Tetrahedron Lett. 26:647-650; andDiMaio et al. (1989) J. Chem. Soc. Perkin Trans. p. 1687; a Gly-Ala turnanalog (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bondisostere (Clones et al. (1988) Tetrahedron Lett. 29:3853-3856);tetrazole (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC(Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501:509); andanalogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 andGarvey et al. (1990) J. Org. Chem. 56:436. Conformationally restrictedmimetics of beta turns and beta bulges, and peptides containing them,are described in U.S. Pat. No. 5,440,013, issued Aug. 8, 1995 to Kahn.

It is known to those skilled in the art that modifications can be madeto any peptide by substituting one or more amino acids with one or morefunctionally equivalent amino acids that does not alter the biologicalfunction of the peptide. In one aspect, the amino acid that issubstituted by an amino acid that possesses similar intrinsic propertiesincluding, but not limited to, hydrophobicity, size, or charge. Methodsused to determine the appropriate amino acid to be substituted and forwhich amino acid are known to one of skill in the art. Non-limitingexamples include empirical substitution models as described by Dahoff etal. (1978) In Atlas of Protein Sequence and Structure Vol. 5 suppl. 2(ed. M. O. Dayhoff), pp. 345-352. National Biomedical ResearchFoundation, Washington D.C.; PAM matrices including Dayhoff matrices(Dahoff et al. (1978), supra, or JTT matrices as described by Jones etal. (1992) Comput. Appl. Biosci. 8:275-282 and Gonnet et al. (1992)Science 256:1443-1145; the empirical model described by Adach andHasegawa (1996) J. Mol. Evol. 42:459-468; the block substitutionmatrices (BLOSUM) as described by Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89:10915-10919; Poisson models as described by Nei(1987) Molecular Evolutionary Genetics. Columbia University Press, NewYork; and the Maximum Likelihood (ML) Method as described by Müller etal. (2002) Mol. Biol. Evol. 19:8-13.

Polypeptide Conjugates

The polypeptides and polypeptide complexes of the invention can be usedin a variety of formulations, which may vary depending on the intendeduse. For example, one or more can be covalently or non-covalently linked(complexed) to various other molecules, the nature of which may varydepending on the particular purpose. For example, a peptide of theinvention can be covalently or non-covalently complexed to amacromolecular carrier, including, but not limited to, natural andsynthetic polymers, proteins, polysaccharides, polypeptides (aminoacids), polyvinyl alcohol, polyvinyl pyrrolidone, and lipids. A peptidecan be conjugated to a fatty acid, for introduction into a liposome, seeU.S. Pat. No. 5,837,249. A peptide of the invention can be complexedcovalently or non-covalently with a solid support, a variety of whichare known in the art and described herein. An antigenic peptide epitopeof the invention can be associated with an antigen-presenting matrixsuch as an MHC complex with or without co-stimulatory molecules.

Examples of protein carriers include, but are not limited to,superantigens, serum albumin, tetanus toxoid, ovalbumin, thyroglobulin,myoglobulin, and immunoglobulin.

Peptide-protein carrier polymers may be formed using conventionalcross-linking agents such as carbodimides. Examples of carbodimides are1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide (CMC),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and1-ethyl-3-(4-azonia-44-dimethylpentyl) carbodiimide.

Examples of other suitable cross-linking agents are cyanogen bromide,glutaraldehyde and succinic anhydride. In general, any of a number ofhomo-bifunctional agents including a homo-bifunctional aldehyde, ahomo-bifunctional epoxide, a homo-bifunctional imido-ester, ahomo-bifunctional N-hydroxysuccinimide ester, a homo-bifunctionalmaleimide, a homo-bifunctional alkyl halide, a homo-bifunctional pyridyldisulfide, a homo-bifunctional aryl halide, a homo-bifunctionalhydrazide, a homo-bifunctional diazonium derivative and ahomo-bifunctional photoreactive compound may be used. Also included arehetero-bifunctional compounds, for example, compounds having anamine-reactive and a sulfhydryl-reactive group, compounds with anamine-reactive and a photoreactive group and compounds with acarbonyl-reactive and a sulfhydryl-reactive group.

Specific examples of such homo-bifunctional cross-linking agents includethe bifunctional N-hydroxysuccinimide estersdithiobis(succinimidylpropionate), disuccinimidyl suberate, anddisuccinimidyl tartrate; the bifunctional imido-esters dimethyladipimidate, dimethyl pimelimidate, and dimethyl suberimidate; thebifunctional sulfhydryl-reactive crosslinkers1,4-di-[3′-(2′-pyridyldithio) propionamido]butane, bismaleimidohexane,and bis-N-maleimido-1,8-octane; the bifunctional aryl halides1,5-difluoro-2,4-dinitrobenzene and4,4′-difluoro-3,3′-dinitrophenylsulfone; bifunctional photoreactiveagents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide; thebifunctional aldehydes formaldehyde, malondialdehyde, succinaldehyde,glutaraldehyde, and adipaldehyde; a bifunctional epoxide such as1,4-butaneodiol diglycidyl ether; the bifunctional hydrazides adipicacid dihydrazide, carbohydrazide, and succinic acid dihydrazide; thebifunctional diazoniums o-tolidine, diazotized and bis-diazotizedbenzidine; the bifunctional alkylhalidesN1N′-ethylene-bis(iodoacetamide), N1N′-hexamethylene-bis(iodoacetamide),N1N′-undecamethylene-bis(iodoacetamide), as well as benzylhalides andhalomustards, such as a1a′-diiodo-p-xylene sulfonic acid andtri(2-chloroethyl)amine, respectively.

Examples of common hetero-bifunctional cross-linking agents that may beused to effect the conjugation of proteins to peptides include, but arenot limited to, SMCC(succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS(m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB(N-succinimidyl(4-iodoacteyl)aminobenzoate), SMPB(succinimidyl-4-(p-maleimidophenyl)butyrate), GMBS(N-(γ-maleimidobutyryloxy)succinimide ester), MPBH(4-(4-N-maleimidopohenyl) butyric acid hydrazide), M2C2H(4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide), SMPT(succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), and SPDP(N-succinimidyl 3-(2-pyridyldithio)propionate).

Cross-linking may be accomplished by coupling a carbonyl group to anamine group or to a hydrazide group by reductive amination.

Peptides of the invention also may be formulated as non-covalentattachment of monomers through ionic, adsorptive, or biospecificinteractions. Complexes of peptides with highly positively or negativelycharged molecules may be done through salt bridge formation under lowionic strength environments, such as in deionized water. Large complexescan be created using charged polymers such as poly-(L-glutamic acid) orpoly-(L-lysine) which contain numerous negative and positive charges,respectively. Adsorption of peptides may be done to surfaces such asmicroparticle latex beads or to other hydrophobic polymers, formingnon-covalently associated peptide-superantigen complexes effectivelymimicking cross-linked or chemically polymerized protein. Finally,peptides may be non-covalently linked through the use of biospecificinteractions between other molecules. For instance, utilization of thestrong affinity of biotin for proteins such as avidin or streptavidin ortheir derivatives could be used to form peptide complexes. Thesebiotin-binding proteins contain four binding sites that can interactwith biotin in solution or be covalently attached to another molecule.(See Wilchek (1988) Anal. Biochem. 171:1-32). Peptides can be modifiedto possess biotin groups using common biotinylation reagents such as theN-hydroxysuccinimidyl ester of D-biotin (NHS-biotin) which reacts withavailable amine groups on the protein. Biotinylated peptides then can beincubated with avidin or streptavidin to create large complexes. Themolecular mass of such polymers can be regulated through careful controlof the molar ratio of biotinylated peptide to avidin or streptavidin.

Also provided by this application are the peptides and polypeptidesdescribed herein conjugated to a label, e.g., a fluorescent orbioluminescent label, for use in the diagnostic methods. For example,detectably labeled peptides and polypeptides can be bound to a columnand used for the detection and purification of antibodies. Suitablefluorescent labels include, but are not limited to, fluorescein,rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow,Cascade Blue™, and Texas Red. Other suitable optical dyes are describedin Haugland, Richard P. (1996) Molecular Probes Handbook.

The polypeptides of this invention also can be combined with variousliquid phase carriers, such as sterile or aqueous solutions,pharmaceutically acceptable carriers, suspensions and emulsions.Examples of non-aqueous solvents include propyl ethylene glycol,polyethylene glycol and vegetable oils. When used to prepare antibodies,the carriers also can include an adjuvant that is useful tonon-specifically augment a specific immune response. A skilled artisancan easily determine whether an adjuvant is required and select one.However, for the purpose of illustration only, suitable adjuvantsinclude, but are not limited to, Freund's Complete Adjuvant, Freund'sIncomplete Adjuvant and mineral salts.

Host Cells

Also provided are host cells comprising one or more of the polypeptidesof this invention. In one aspect, the polypeptides are expressed andpresent on the cell surface (extracellularly). Suitable cells containingthe inventive polypeptides include prokaryotic and eukaryotic cells,which include, but are not limited to bacterial cells, yeast cells,insect cells, animal cells, mammalian cells, murine cells, rat cells,sheep cells, simian cells and human cells. Examples of bacterial cellsinclude Escherichia coli, Salmonella enterica and Streptococcusgordonii. The cells can be purchased from a commercial vendor such asthe American Type Culture Collection (ATCC, Rockville Md., USA) orcultured from an isolate using methods known in the art. Examples ofsuitable eukaryotic cells include, but are not limited to 293T HEKcells, as well as the hamster cell line CHO, BHK-21; the murine celllines designated NIH3T3, NS0, C127, the simian cell lines COS, Vero; andthe human cell lines HeLa, PER.C6 (commercially available from Crucell)U-937 and Hep G2. A non-limiting example of insect cells includeSpodoptera frugiperda. Examples of yeast useful for expression include,but are not limited to Saccharomyces, Schizosaccharomyces, Hansenula,Candida, Torulopsis, Yarrowia, or Pichia. See e.g., U.S. Pat. Nos.4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and6,258,559.

In addition to species specificity, the cells can be of any particulartissue type such as neuronal or alternatively a somatic or embryonicstem cell such as a stem cell that can or cannot differentiate into aneuronal cell, e.g., embryonic stem cell, adipose stem cell, neuronalstem cell and hematopoietic stem cell. The stem cell can be of human oranimal origin, such as mammalian.

Isolated Polynucleotides and Compositions

This invention also provides the complementary polynucleotides to thesequences identified above or their complements. Complementarity can bedetermined using traditional hybridization under conditions of moderateor high stringency. As used herein, the term polynucleotide intends DNAand RNA as well as modified nucleotides. For example, this inventionalso provides the anti-sense polynucleotide stand, e.g. antisense RNA tothese sequences or their complements.

Also provided are polynucleotides encoding substantially homologous andbiologically equivalent polypeptides to the inventive polypeptides andpolypeptide complexes. Substantially homologous and biologicallyequivalent intends those having varying degrees of homology, such as atleast 65%, or alternatively, at least 70%, or alternatively, at least75%, or alternatively at least 80%, or alternatively, at least 85%, oralternatively at least 90%, or alternatively, at least 95%, oralternatively at least 97% homologous as defined above and which encodepolypeptides having the biological activity to bind factor Xa inhibitorsand do not assemble into the prothrombinase complex as described herein.It should be understood although not always explicitly stated thatembodiments to substantially homologous polypeptides and polynucleotidesare intended for each aspect of this invention, e.g., polypeptides,polynucleotides and antibodies.

The polynucleotides of this invention can be replicated usingconventional recombinant techniques. Alternatively, the polynucleotidescan be replicated using PCR technology. PCR is the subject matter ofU.S. Pat. Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202 anddescribed in PCR: The Polymerase Chain Reaction (Mullis et al. eds,Birkhauser Press, Boston (1994)) and references cited therein. Yetfurther, one of skill in the art can use the sequences provided hereinand a commercial DNA synthesizer to replicate the DNA. Accordingly, thisinvention also provides a process for obtaining the polynucleotides ofthis invention by providing the linear sequence of the polynucleotide,appropriate primer molecules, chemicals such as enzymes and instructionsfor their replication and chemically replicating or linking thenucleotides in the proper orientation to obtain the polynucleotides. Ina separate embodiment, these polynucleotides are further isolated. Stillfurther, one of skill in the art can operatively link thepolynucleotides to regulatory sequences for their expression in a hostcell. The polynucleotides and regulatory sequences are inserted into thehost cell (prokaryotic or eukaryotic) for replication and amplification.The DNA so amplified can be isolated from the cell by methods well knownto those of skill in the art. A process for obtaining polynucleotides bythis method is further provided herein as well as the polynucleotides soobtained.

RNA can be obtained by first inserting a DNA polynucleotide into asuitable prokaryotic or eukaryotic host cell. The DNA can be inserted byany appropriate method, e.g., by the use of an appropriate gene deliveryvehicle (e.g., liposome, plasmid or vector) or by electroporation. Whenthe cell replicates and the DNA is transcribed into RNA; the RNA canthen be isolated using methods well known to those of skill in the art,for example, as set forth in Sambrook and Russell (2001) supra. Forinstance, mRNA can be isolated using various lytic enzymes or chemicalsolutions according to the procedures set forth in Sambrook and Russell(2001) supra or extracted by nucleic-acid-binding resins following theaccompanying instructions provided by manufactures.

In one aspect, the RNA is short interfering RNA, also known as siRNA.Methods to prepare and screen interfering RNA and select for the abilityto block polynucleotide expression are known in the art and non-limitingexamples of which are shown below. These interfering RNA are provided bythis invention.

siRNA sequences can be designed by obtaining the target mRNA sequenceand determining an appropriate siRNA complementary sequence. siRNAs ofthe invention are designed to interact with a target sequence, meaningthey complement a target sequence sufficiently to hybridize to thatsequence. An siRNA can be 100% identical to the target sequence.However, homology of the siRNA sequence to the target sequence can beless than 100% as long as the siRNA can hybridize to the targetsequence. Thus, for example, the siRNA molecule can be at least 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to thetarget sequence or the complement of the target sequence. Therefore,siRNA molecules with insertions, deletions or single point mutationsrelative to a target may also be used. The generation of severaldifferent siRNA sequences per target mRNA is recommended to allowscreening for the optimal target sequence. A homology search, such as aBLAST search, should be performed to ensure that the siRNA sequence doesnot contain homology to any known mammalian gene.

In general, it is preferable that the target sequence be located atleast 100-200 nucleotides from the AUG initiation codon and at least50-100 nucleotides away from the termination codon of the target mRNA(Duxbury (2004) J. Surgical Res. 117:339-344).

Researchers have determined that certain characteristics are common insiRNA molecules that effectively silence their target gene (Duxbury(2004) J. Surgical Res. 117:339-344; Ui-Tei et al. (2004) Nucl. AcidsRes. 32:936-48). As a general guide, siRNAs that include one or more ofthe following conditions are particularly useful in gene silencing inmammalian cells: GC ratio of between 45-55%, no runs of more than 9 G/Cresidues, G/C at the 5′ end of the sense strand; A/U at the 5′ end ofthe antisense strand; and at least 5 A/U residues in the first 7 basesof the 5′ terminal of the antisense strand.

siRNA are, in general, from about 10 to about 30 nucleotides in length.For example, the siRNA can be 10-30 nucleotides long, 12-28 nucleotideslong, 15-25 nucleotides long, 19-23 nucleotides long, or 21-23nucleotides long. When an siRNA contains two strands of differentlengths, the longer of the strands designates the length of the siRNA.In this situation, the unpaired nucleotides of the longer strand wouldform an overhang.

The term siRNA includes short hairpin RNAs (shRNAs). shRNAs comprise asingle strand of RNA that forms a stem-loop structure, where the stemconsists of the complementary sense and antisense strands that comprisea double-stranded siRNA, and the loop is a linker of varying size. Thestem structure of shRNAs generally is from about 10 to about 30nucleotides long. For example, the stem can be 10-30 nucleotides long,12-28 nucleotides long, 15-25 nucleotides long, 19-23 nucleotides long,or 21-23 nucleotides long.

Tools to assist siRNA design are readily available to the public. Forexample, a computer-based siRNA design tool is available on the internetat www.dharmacon.com, last accessed on Nov. 26, 2007.

Synthesis of dsRNA and siRNA

dsRNA and siRNA can be synthesized chemically or enzymatically in vitroas described in Micura (2002) Agnes Chem. Int. Ed. Emgl. 41:2265-2269;Betz (2003) Promega Notes 85:15-18; and Paddison and Hannon (2002)Cancer Cell. 2:17-23. Chemical synthesis can be performed via manual orautomated methods, both of which are well known in the art as describedin Micura (2002), supra. siRNA can also be endogenously expressed insidethe cells in the form of shRNAs as described in Yu et al. (2002) Proc.Natl. Acad. Sci. USA 99:6047-6052; and McManus et al. (2002) RNA8:842-850. Endogenous expression has been achieved using plasmid-basedexpression systems using small nuclear RNA promoters, such as RNApolymerase III U6 or H1, or RNA polymerase II U1 as described inBrummelkamp et al. (2002) Science 296:550-553 (2002); and Novarino etal. (2004) J. Neurosci. 24:5322-5330.

In vitro enzymatic dsRNA and siRNA synthesis can be performed using anRNA polymerase mediated process to produce individual sense andantisense strands that are annealed in vitro prior to delivery into thecells of choice as describe in Fire et al. (1998) Nature 391:806-811;Donze and Picard (2002) Nucl. Acids Res. 30(10):e46; Yu et al. (2002);and Shim et al. (2002) J. Biol. Chem. 277:30413-30416. Severalmanufacturers (Promega, Ambion, New England Biolabs, and Stragene)produce transcription kits useful in performing the in vitro synthesis.

In vitro synthesis of siRNA can be achieved, for example, by using apair of short, duplex oligonucleotides that contain T7 RNA polymerasepromoters upstream of the sense and antisense RNA sequences as the DNAtemplate. Each oligonucleotide of the duplex is a separate template forthe synthesis of one strand of the siRNA. The separate short RNA strandsthat are synthesized are then annealed to form siRNA as described inProtocols and Applications, Chapter 2: RNA interference, PromegaCorporation, (2005).

In vitro synthesis of dsRNA can be achieved, for example, by using a T7RNA polymerase promoter at the 5′-ends of both DNA target sequencestrands. This is accomplished by using separate DNA templates, eachcontaining the target sequence in a different orientation relative tothe T7 promoter, transcribed in two separate reactions. The resultingtranscripts are mixed and annealed post-transcriptionally. DNA templatesused in this reaction can be created by PCR or by using two linearizedplasmid templates, each containing the T7 polymerase promoter at adifferent end of the target sequence. Protocols and Applications,Chapter 2: RNA interference, Promega Corporation, (2005).

In order to express the proteins described herein, delivery of nucleicacid sequences encoding the gene of interest can be delivered by severaltechniques. Examples of which include viral technologies (e.g.retroviral vectors, adenovirus vectors, adeno-associated virus vectors,alphavirus vectors and the like) and non-viral technologies (e.g.DNA/liposome complexes, micelles and targeted viral protein-DNAcomplexes) as described herein. Once inside the cell of interest,expression of the transgene can be under the control of ubiquitouspromoters (e.g. EF-1α) or tissue specific promoters (e.g. CalciumCalmodulin kinase 2 (CaMKI) promoter, NSE promoter and human Thy-1promoter). Alternatively expression levels may controlled by use of aninducible promoter system (e.g. Tet on/off promoter) as described inWiznerowicz et al. (2005) Stem Cells 77:8957-8961.

Non-limiting examples of promoters include, but are not limited to, thecytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet.8:148-154), CMV/human β3-globin promoter (Mandel et al. (1998) J.Neurosci. 18:4271-4284), NCX1 promoter, αMHC promoter, MLC2v promoter,GFAP promoter (Xu et al. (2001) Gene Ther., 8:1323-1332), the 1.8-kbneuron-specific enolase (NSE) promoter (Klein et al. (1998) Exp. Neurol.150:183-194), chicken beta actin (CBA) promoter (Miyazaki (1989) Gene79:269-277) and the β-glucuronidase (GUSB) promoter (Shipley et al.(1991) Genetics 10:1009-1018), the human serum albumin promoter, thealpha-1-antitrypsin promoter. To improve expression, other regulatoryelements may additionally be operably linked to the transgene, such as,e.g., the Woodchuck Hepatitis Virus Post-Regulatory Element (WPRE)(Donello et al. (1998) J. Virol. 72: 5085-5092) or the bovine growthhormone (BGH) polyadenylation site.

Also provided herein is a polynucleotide probe or primer comprising atleast 10, or alternatively, at least 17 or alternatively at least 20, oralternatively, at least 50, or alternatively, at least 75polynucleotides, or alternatively at least 100 polynucleotides encodingSEQ ID NOS: 12 through 15 or their complements. Suitable probes andprimers are described supra. It is known in the art that a “perfectlymatched” probe is not needed for a specific hybridization. Minor changesin probe sequence achieved by substitution, deletion or insertion of asmall number of bases do not affect the hybridization specificity. Ingeneral, as much as 20% base-pair mismatch (when optimally aligned) canbe tolerated. A probe useful for detecting the aforementioned mRNA is atleast about 80% identical to the homologous region of comparable sizecontained in the previously identified sequences (identified above)which correspond to previously characterized polynucleotides of thisinvention. Alternatively, the probe is 85% identical to thecorresponding gene sequence after alignment of the homologous region;and yet further, it exhibits 90% identity, or still further, at least95% identical.

These probes can be used in radioassays (e.g. Southern and Northern blotanalysis) to detect or monitor expression of the polynucleotides orpolypeptides of this invention. The probes also can be attached to asolid support or an array such as a chip for use in high throughputscreening assays for the detection of expression of the genecorresponding to one or more polynucleotide(s) of this invention.

The polynucleotides and fragments of the polynucleotides of the presentinvention also can serve as primers for the detection of genes or genetranscripts that are expressed in neuronal cells, for example, toconfirm transduction of the polynucleotides into host cells. In thiscontext, amplification means any method employing a primer-dependentpolymerase capable of replicating a target sequence with reasonablefidelity. Amplification may be carried out by natural or recombinantDNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coliDNA polymerase, and reverse transcriptase. Primer length is the same asthat identified for probes, above.

The invention further provides the isolated polynucleotides of thisinvention operatively linked to a promoter of RNA transcription, as wellas other regulatory sequences for replication and/or transient or stableexpression of the DNA or RNA. As used herein, the term “operativelylinked” means positioned in such a manner that the promoter will directtranscription of RNA off the DNA molecule. Examples of such promotersare SP6, T4 and T7. In certain embodiments, cell-specific promoters areused for cell-specific expression of the inserted polynucleotide.Vectors which contain a promoter or a promoter/enhancer, withtermination codons and selectable marker sequences, as well as a cloningsite into which an inserted piece of DNA can be operatively linked tothat promoter are well known in the art and commercially available. Forgeneral methodology and cloning strategies, see Gene ExpressionTechnology (Goeddel ed., Academic Press, Inc. (1991)) and referencescited therein and Vectors: Essential Data Series (Gacesa and Ramji,eds., John Wiley & Sons, N.Y. (1994)), which contains maps, functionalproperties, commercial suppliers and a reference to GenEMBL accessionnumbers for various suitable vectors. Preferable, these vectors arecapable of transcribing RNA in vitro or in vivo.

Expression vectors containing these nucleic acids are useful to obtainhost vector systems to produce proteins and polypeptides. It is impliedthat these expression vectors must be replicable in the host organismseither as episomes or as an integral part of the chromosomal DNA.Suitable expression vectors include plasmids, viral vectors, includingadenoviruses, adeno-associated viruses, retroviruses, cosmids, etc.Adenoviral vectors are particularly useful for introducing genes intotissues in vivo because of their high levels of expression and efficienttransformation of cells both in vitro and in vivo. When a nucleic acidis inserted into a suitable host cell, e.g., a prokaryotic or aeukaryotic cell and the host cell replicates, the protein can berecombinantly produced. Suitable host cells will depend on the vectorand can include mammalian cells, animal cells, human cells, simiancells, insect cells, yeast cells, and bacterial cells as described aboveand constructed using well known methods. See Sambrook and Russell(2001), supra. In addition to the use of viral vector for insertion ofexogenous nucleic acid into cells, the nucleic acid can be inserted intothe host cell by methods well known in the art such as transformationfor bacterial cells; transfection using calcium phosphate precipitationfor mammalian cells; DEAE-dextran; electroporation; or microinjection.See Sambrook and Russell (2001), supra for this methodology.

The present invention also provides delivery vehicles suitable fordelivery of a polynucleotide of the invention into cells (whether invivo, ex vivo, or in vitro). A polynucleotide of the invention can becontained within a gene delivery vehicle, a cloning vector or anexpression vector. These vectors (especially expression vectors) can inturn be manipulated to assume any of a number of forms which may, forexample, facilitate delivery to and/or entry into a cell.

These isolated host cells containing the polynucleotides of thisinvention are useful for the recombinant replication of thepolynucleotides and for the recombinant production of peptides and forhigh throughput screening.

The polynucleotides of this invention can be conjugated to a detectablelabel or combined with a carrier such as a solid support orpharmaceutically acceptable carrier. Suitable solid supports aredescribed above as well as have suitable labels. Methods for attaching alabel to a polynucleotide are known to those skilled in the art. SeeSambrook and Russell (2001), supra.

Therapeutic Antibody Compositions

This invention also provides an antibody capable of specifically forminga complex with a protein or polypeptide of this invention, which areuseful in the therapeutic methods of this invention. The term “antibody”includes polyclonal antibodies and monoclonal antibodies, antibodyfragments, as well as derivatives thereof (described above). Theantibodies include, but are not limited to mouse, rat, and rabbit orhuman antibodies. Antibodies can be produced in cell culture, in phage,or in various animals, including but not limited to cows, rabbits,goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys,chimpanzees, apes, etc. The antibodies are also useful to identify andpurify therapeutic polypeptides.

This invention also provides an antibody-peptide complex comprisingantibodies described above and a polypeptide that specifically binds tothe antibody. In one aspect the polypeptide is the polypeptide againstwhich the antibody was raised. In one aspect the antibody-peptidecomplex is an isolated complex. In a further aspect, the antibody of thecomplex is, but not limited to, a polyclonal antibody, a monoclonalantibody, a humanized antibody or an antibody derivative describedherein. Either or both of the antibody or peptide of theantibody-peptide complex can be detectably labeled. In one aspect, theantibody-peptide complex of the invention can be used as a control orreference sample in diagnostic or screening assays.

Polyclonal antibodies of the invention can be generated usingconventional techniques known in the art and are well-described in theliterature. Several methodologies exist for production of polyclonalantibodies. For example, polyclonal antibodies are typically produced byimmunization of a suitable mammal such as, but not limited to, chickens,goats, guinea pigs, hamsters, horses, mice, rats, and rabbits. Anantigen is injected into the mammal, which induces the B-lymphocytes toproduce IgG immunoglobulins specific for the antigen. This IgG ispurified from the mammals serum. Variations of this methodology includemodification of adjuvants, routes and site of administration, injectionvolumes per site and the number of sites per animal for optimalproduction and humane treatment of the animal. For example, adjuvantstypically are used to improve or enhance an immune response to antigens.Most adjuvants provide for an injection site antiben depot, which allowsfor a slow release of antigen into draining lymph nodes. Other adjuvantsinclude surfactants which promote concentration of protein antigenmolecules over a large surface area and immunostimulatory molecules.Non-limiting examples of adjuvants for polyclonal antibody generationinclude Freund's adjuvants, Ribi adjuvant system, and Titermax.Polyclonal antibodies can be generated using methods described in U.S.Pat. Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746;6,322,788; 5,686,073; and 5,670,153.

The monoclonal antibodies of the invention can be generated usingconventional hybridoma techniques known in the art and well-described inthe literature. For example, a hybridoma is produced by fusing asuitable immortal cell line (e.g., a myeloma cell line such as, but notlimited to, Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, >243,P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U397, MLA 144, ACT IV,MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144,NAMAIWA, NEURO 2A, CHO, PerC.6, YB2/O) or the like, or heteromyelomas,fusion products thereof, or any cell or fusion cell derived therefrom,or any other suitable cell line as known in the art (see, e.g.,www.atcc.org, www.lifetech.com, last accessed on Nov. 26, 2007, and thelike), with antibody producing cells, such as, but not limited to,isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. Antibodyproducing cells can also be obtained from the peripheral blood or,preferably the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing-heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods.

In one embodiment, the antibodies described herein can be generatedusing a Multiple Antigenic Peptide (MAP) system. The MAP system utilizesa peptidyl core of three or seven radially branched lysine residues, onto which the antigen peptides of interest can be built using standardsolid-phase chemistry. The lysine core yields the MAP bearing about 4 to8 copies of the peptide epitope depending on the inner core thatgenerally accounts for less than 10% of total molecular weight. The MAPsystem does not require a carrier protein for conjugation. The highmolar ratio and dense packing of multiple copies of the antigenicepitope in a MAP has been shown to produce strong immunogenic response.This method is described in U.S. Pat. No. 5,229,490 and is hereinincorporated by reference in its entirety.

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from various commercial vendors such as Cambridge AntibodyTechnologies (Cambridgeshire, UK), MorphoSys (Martinsreid/Planegg,Del.), Biovation (Aberdeen, Scotland, UK) BioInvent (Lund, Sweden),using methods known in the art. See U.S. Pat. Nos. 4,704,692; 5,723,323;5,763,192; 5,814,476; 5,817,483; 5,824,514; 5,976,862. Alternativemethods rely upon immunization of transgenic animals (e.g., SCID mice,Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu etal. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol.93:154-161 that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal. (1997) Proc. Natl. Acad. Sci. USA, 94:4937-4942; Hanes et al. (1998)Proc. Natl. Acad. Sci. USA, 95:14130-14135); single cell antibodyproducing technologies (e.g., selected lymphocyte antibody method(“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al. (1987) J. Immunol.17:887-892; Babcook et al., Proc. Natl. Acad. Sci. USA (1996)93:7843-7848); gel microdroplet and flow cytometry (Powell et al. (1990)Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass.); Gray et al.(1995) J. Imm. Meth. 182:155-163; and Kenny et al. (1995) Bio. Technol.13:787-790); B-cell selection (Steenbakkers et al. (1994) Molec. Biol.Reports 19:125-134.

Antibody derivatives of the present invention can also be prepared bydelivering a polynucleotide encoding an antibody of this invention to asuitable host such as to provide transgenic animals or mammals, such asgoats, cows, horses, sheep, and the like, that produce such antibodiesin their milk. These methods are known in the art and are described forexample in U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992;5,994,616; 5,565,362; and 5,304,489.

The term “antibody derivative” includes post-translational modificationto linear polypeptide sequence of the antibody or fragment. For example,U.S. Pat. No. 6,602,684 B1 describes a method for the generation ofmodified glycol-forms of antibodies, including whole antibody molecules,antibody fragments, or fusion proteins that include a region equivalentto the Fc region of an immunoglobulin, having enhanced Fc-mediatedcellular toxicity, and glycoproteins so generated.

Antibody derivatives also can be prepared by delivering a polynucleotideof this invention to provide transgenic plants and cultured plant cells(e.g., but not limited to tobacco, maize, and duckweed) that producesuch antibodies, specified portions or variants in the plant parts or incells cultured therefrom. For example, Cramer et al. (1999) Curr. Top.Microbol. Immunol. 240:95-118 and references cited therein, describe theproduction of transgenic tobacco leaves expressing large amounts ofrecombinant proteins, e.g., using an inducible promoter. Transgenicmaize have been used to express mammalian proteins at commercialproduction levels, with biological activities equivalent to thoseproduced in other recombinant systems or purified from natural sources.See, e.g., Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 andreferences cited therein. Antibody derivatives have also been producedin large amounts from transgenic plant seeds including antibodyfragments, such as single chain antibodies (scFv's), including tobaccoseeds and potato tubers. See, e.g., Conrad et al. (1998) Plant Mol.Biol. 38:101-109 and reference cited therein. Thus, antibodies of thepresent invention can also be produced using transgenic plants,according to know methods.

Antibody derivatives also can be produced, for example, by addingexogenous sequences to modify immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic. Generally part or allof the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions are replacedwith human or other amino acids.

In general, the CDR residues are directly and most substantiallyinvolved in influencing antigen binding. Humanization or engineering ofantibodies of the present invention can be performed using any knownmethod such as, but not limited to, those described in U.S. Pat. Nos.5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192;5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762;5,530,101; 5,585,089; 5,225,539; and 4,816,567.

Techniques for making partially to fully human antibodies are known inthe art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice which are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. (See for example, Russel et al. (2000) Infection and ImmunityApril 2000:1820-1826; Gallo et al. (2000) European J. of Immun.30:534-540; Green (1999) J. of Immun. Methods 231:11-23; Yang et al.(1999A) J. of Leukocyte Biology 66:401-410; Yang (1999B) Cancer Research59(6):1236-1243; Jakobovits. (1998) Advanced Drug Delivery Reviews31:33-42; Green and Jakobovits (1998) J. Exp. Med. 188(3):483-495;Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4):607-614; Tsuda et al.(1997) Genomics 42:413-421; Sherman-Gold (1997) Genetic Engineering News17(14); Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits(1996) Weir's Handbook of Experimental Immunology, The Integrated ImmuneSystem Vol. IV, 194.1-194.7; Jakobovits (1995) Current Opinion inBiotechnology 6:561-566; Mendez et al. (1995) Genomics 26:294-307;Jakobovits (1994) Current Biology 4(8):761-763; Arbones et al. (1994)Immunity 1(4):247-260; Jakobovits (1993) Nature 362(6417):255-258;Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA 90(6):2551-2555; andU.S. Pat. No. 6,075,181.)

The antibodies of this invention also can be modified to create chimericantibodies. Chimeric antibodies are those in which the various domainsof the antibodies' heavy and light chains are coded for by DNA from morethan one species. See, e.g., U.S. Pat. No. 4,816,567.

Alternatively, the antibodies of this invention can also be modified tocreate veneered antibodies. Veneered antibodies are those in which theexterior amino acid residues of the antibody of one species arejudiciously replaced or “veneered” with those of a second species sothat the antibodies of the first species will not be immunogenic in thesecond species thereby reducing the immunogenicity of the antibody.Since the antigenicity of a protein is primarily dependent on the natureof its surface, the immunogenicity of an antibody could be reduced byreplacing the exposed residues which differ from those usually found inanother mammalian species antibodies. This judicious replacement ofexterior residues should have little, or no, effect on the interiordomains, or on the interdomain contacts. Thus, ligand binding propertiesshould be unaffected as a consequence of alterations which are limitedto the variable region framework residues. The process is referred to as“veneering” since only the outer surface or skin of the antibody isaltered, the supporting residues remain undisturbed.

The procedure for “veneering” makes use of the available sequence datafor human antibody variable domains compiled by Kabat et al. (1987)Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md.,National Institutes of Health, updates to this database, and otheraccessible U.S. and foreign databases (both nucleic acid and protein).Non-limiting examples of the methods used to generate veneeredantibodies include EP 519596; U.S. Pat. No. 6,797,492; and described inPadlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

The term “antibody derivative” also includes “diabodies” which are smallantibody fragments with two antigen-binding sites, wherein fragmentscomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) in the same polypeptide chain. (See for example, EP404,097; WO 93/11161; and Hollinger et al., (1993) Proc. Natl. Acad.Sci. USA 90:6444-6448.) By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. (See also, U.S. Pat. No. 6,632,926 toChen et al. which discloses antibody variants that have one or moreamino acids inserted into a hypervariable region of the parent antibodyand a binding affinity for a target antigen which is at least about twofold stronger than the binding affinity of the parent antibody for theantigen.)

The term “antibody derivative” further includes “linear antibodies”. Theprocedure for making linear antibodies is known in the art and describedin Zapata et al. (1995) Protein Eng. 8(10):1057-1062. Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-VH-C_(H)1) which form a pair of antigen binding regions.Linear antibodies can be bispecific or monospecific.

The antibodies of this invention can be recovered and purified fromrecombinant cell cultures by known methods including, but not limitedto, protein A purification, ammonium sulfate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe used for purification.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells, or alternatively from aprokaryotic cells as described above.

If a monoclonal antibody being tested binds with protein or polypeptide,then the antibody being tested and the antibodies provided by thehybridomas of this invention are equivalent. It also is possible todetermine without undue experimentation, whether an antibody has thesame specificity as the monoclonal antibody of this invention bydetermining whether the antibody being tested prevents a monoclonalantibody of this invention from binding the protein or polypeptide withwhich the monoclonal antibody is normally reactive. If the antibodybeing tested competes with the monoclonal antibody of the invention asshown by a decrease in binding by the monoclonal antibody of thisinvention, then it is likely that the two antibodies bind to the same ora closely related epitope. Alternatively, one can pre-incubate themonoclonal antibody of this invention with a protein with which it isnormally reactive, and determine if the monoclonal antibody being testedis inhibited in its ability to bind the antigen. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or a closely related, epitopic specificity as the monoclonalantibody of this invention.

The term “antibody” also is intended to include antibodies of allisotypes. Particular isotypes of a monoclonal antibody can be preparedeither directly by selecting from the initial fusion, or preparedsecondarily, from a parental hybridoma secreting a monoclonal antibodyof different isotype by using the sib selection technique to isolateclass switch variants using the procedure described in Steplewski, etal. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira, et al. (1984) J.Immunol. Methods 74:307.

The isolation of other hybridomas secreting monoclonal antibodies withthe specificity of the monoclonal antibodies of the invention can alsobe accomplished by one of ordinary skill in the art by producinganti-idiotypic antibodies. Herlyn, et al. (1986) Science 232:100. Ananti-idiotypic antibody is an antibody which recognizes uniquedeterminants present on the monoclonal antibody produced by thehybridoma of interest.

Idiotypic identity between monoclonal antibodies of two hybridomasdemonstrates that the two monoclonal antibodies are the same withrespect to their recognition of the same epitopic determinant. Thus, byusing antibodies to the epitopic determinants on a monoclonal antibodyit is possible to identify other hybridomas expressing monoclonalantibodies of the same epitopic specificity.

It is also possible to use the anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is themirror image of the epitope bound by the first monoclonal antibody.Thus, in this instance, the anti-idiotypic monoclonal antibody could beused for immunization for production of these antibodies.

In some aspects of this invention, it will be useful to detectably ortherapeutically label the antibody. Suitable labels are described supra.Methods for conjugating antibodies to these agents are known in the art.For the purpose of illustration only, antibodies can be labeled with adetectable moiety such as a radioactive atom, a chromophore, afluorophore, or the like. Such labeled antibodies can be used fordiagnostic techniques, either in vivo, or in an isolated test sample.

The coupling of antibodies to low molecular weight haptens can increasethe sensitivity of the antibody in an assay. The haptens can then bespecifically detected by means of a second reaction. For example, it iscommon to use haptens such as biotin, which reacts avidin, ordinitrophenol, pyridoxal, and fluorescein, which can react with specificanti-hapten antibodies. See, Harlow and Lane (1988) supra.

Antibodies can be labeled with a detectable moiety such as a radioactiveatom, a chromophore, a fluorophore, or the like. Such labeled antibodiescan be used for diagnostic techniques, either in vivo, or in an isolatedtest sample. Antibodies can also be conjugated, for example, to apharmaceutical agent, such as chemotherapeutic drug or a toxin. They canbe linked to a cytokine, to a ligand, to another antibody. Suitableagents for coupling to antibodies to achieve an anti-tumor effectinclude cytokines, such as interleukin 2 (IL-2) and Tumor NecrosisFactor (TNF); photosensitizers, for use in photodynamic therapy,including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin,and phthalocyanine; radionuclides, such as iodine-131 (¹³¹I), yttrium-90(⁹⁰Y), bismuth-212 (²¹²Bi), bismuth-213 (²¹³Bi), technetium-99m(^(99m)Tc), rhenium-186 (¹⁸⁶Re), and rhenium-188 (¹⁸⁸Re); antibiotics,such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin,neocarzinostatin, and carboplatin; bacterial, plant, and other toxins,such as diphtheria toxin, pseudomonas exotoxin A, staphylococcalenterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and nativericin A), TGF-alpha toxin, cytotoxin from Chinese cobra (naja najaatra), and gelonin (a plant toxin); ribosome inactivating proteins fromplants, bacteria and fungi, such as restrictocin (a ribosomeinactivating protein produced by Aspergillus restrictus), saporin (aribosome inactivating protein from Saponaria officinalis), and RNase;tyrosine kinase inhibitors; ly207702 (a difluorinated purinenucleoside); liposomes containing anti cystic agents (e.g., antisenseoligonucleotides, plasmids which encode for toxins, methotrexate, etc.);and other antibodies or antibody fragments, such as F(ab).

The antibodies of the invention also can be bound to many differentcarriers. Thus, this invention also provides compositions containing theantibodies and another substance, active or inert. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such, using routineexperimentation.

V. Alternative Antidotes and Methods of the Invention

It is contemplated that the unit dose formulation is useful andeffective for antidotes in addition to the polypeptide of SEQ ID NO. 13.

One aspect of the present invention relates to a method of preventing orreducing bleeding in a subject undergoing anticoagulant therapy byadministering to the subject an effective amount of a factor Xa proteinderivative. In one embodiment, the derivative has a modified active siteand/or a modified Gla-domain thereby having either reduced or noprocoagulant activity. The derivative acts as an antidote andsubstantially neutralizes the anticoagulant activity or effect of theinhibitor. In one embodiment, the derivative is either Gla-deficient orGla-domainless. The subject may be a mammal or more particularly, ahuman.

In another embodiment, the invention is directed to a method forselectively binding and inhibiting an exogenously administered factor Xainhibitor in a subject. The method comprises administering to thepatient an effective amount of a derivative of a factor Xa derivative asdescribed above. The subject may be a cell or a mammal, such as a human.

Patients suitable for this therapy have undergone prior anticoagulanttherapy, for example, they have been administered one or more of ananticoagulant, such as an inhibitor of factor Xa. Examples ofanticoagulants that are factor Xa inhibitors, include but are notlimited to, fondaparinux, idraparinux, biotinylated idraparinux,enoxaparin, fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor,DX-9065a, YM-60828, YM-150, apixaban, rivaroxaban, PD-348292,otamixaban, edoxaban, LY517717, GSK913893, low molecular weight heparin,and betrixaban, or any combination thereof. The source of variousanticoagulants is found throughout the description.

In one aspect, the derivative has a modified active site and/or amodified or removed Gla domain. In one aspect, the factor Xa derivativehas or exhibits no procoagulant activity. In this aspect, the derivativecomprises at least amino acid residues 40 to 448, 45 to 448, or 46 to448 of SEQ ID NO. 3 or equivalents thereof. In another aspect, thederivative comprises at least amino acid residues 45 to 139 and 195 to448 or 46 to 139 and 195-448 of SEQ ID NO. 3 or equivalents thereof.

In another aspect of the invention, the fXa derivative retains the threedimensional structure of the active site of the fXa protein. Informationregarding the three-dimensional structure of the des-Gla fXa may befound in Brandstetter, H et al. J. Bio. Chem., 1996, 271:29988-29992.

In another aspect of the invention, the fXa derivatives may lack the Gladomain as well as either one of the two EGF domains. In another aspectof the invention, the fXa derivatives are completely lacking the lightchain. Other modifications of the heavy chain may comprise the catalyticdomain of related serine proteases which are capable of bindinginhibitors. The related serine proteases have catalytic domains whichpossess sufficient structural similarity to fXa catalytic domain and aretherefore capable of binding small molecule fXa inhibitors. Examples ofrelated serine proteases include, but are not limited to, mammalianproteases such as plasma kallikrein, thrombin and trypsin or thebacterial protease subtilisin. These derivatives further includemodifications at the amino acids residues equivalent to the active siteserine (SER379) or aspartic acid (ASP282) residues described herein.

In some embodiments, the factor Xa protein with reduced procoagulantactivity comprises a modified light chain, wherein the modification issubstitution, addition or deletion of the Gla-domain to reduce thephospholipid membrane binding of fXa. In some embodiments, the primeamino acid sequence of fXa is not changed, but the side chain of certainamino acids has been changed. Examples of the modified Gla-domain thatreduces the phospholipid membrane binding of fXa comprises polypeptidesor proteins having the primary amino acid sequence of SEQ ID NO. 3 or anequivalent thereof, with at least one amino acid substitution, addition,or deletion as compared to the Gla-domain of a wild type human factor Xaprotein. In some embodiments, at least one amino acid being substitutedor deleted is a γ-carboxyglutamic acid (Gla). Gla residues are shown inSEQ ID NO. 3 at amino acid positions 6, 7, 14, 16, 19, 20, 25, 26, 29,32, and 39. In some embodiments, the antidote's primary amino acidsequence is identical to SEQ ID NO. 3 or equivalent thereof, but is anuncarboxylated, undercarboxylated or decarboxylated factor Xa protein.In some embodiments, the antidote is a des-Gla anhydro-fXa or des-GlafX-S379A. In some embodiments, the factor Xa protein with reducedphospholipid membrane binding further comprises modification or deletionof the EGF1 and/or EGF2 (shown in FIG. 3 as amino acids 46 to 84 and 85to 128, respectively) or a part, i.e. fragment of the EGF1 and/or EGF2domains. In some embodiments, the entire light chain or substantiallythe entire light chain is modified or removed. For example, the modifiedfXa protein with reduced phospholipid membrane binding may contain onlythe heavy chain or the modified fXa may contain the heavy chain and afragment of the light chain that contains Cys132, the amino acid residuethat forms the single disulfide bond with Cys302 of the heavy chain inSEQ ID NO. 3. In some embodiments, the derivative comprises the aminoacid sequence of SEQ ID NO. 10 or SEQ ID NO. 11. In some embodiments,the derivative is the two chain polypeptide comprising SEQ ID NO. 13. Inother embodiments, the derivative is the polypeptide of SEQ ID NO. 15.

In some embodiments, the factor Xa protein derivative comprises amodified heavy chain that contains the catalytic domain of said factorXa protein. In some embodiments, at least one amino acid substitution ispresent at one or more amino acid position of fXa selected from thegroup consisting of Glu216, Glu218, Arg332, Arg347, Lys351, and Ser379in SEQ ID NOS. 3 and 7 (Glu37, Glu39, Arg150, Arg165, Lys169, and Ser195in chymotrypsin numbering, respectively). In some embodiments, theantidote is a factor Xa protein with active site serine (Ser379 in SEQID NOS. 3 and 7, Ser195 in chymotrypsin numbering) residue modified todehydro-alanine or alanine Such modifications may be made to wild typefXa protein or to any of the modified fXa proteins or fragmentsdescribed above. For example, the des-Gla anhydro-fXa with active siteserine residues replaced by dehydro-alanine described in Example 1 hasshown antidote activity.

In other embodiments, the derivative has reduced interaction with ATIII,cofactors fV/fVa and fVIII/fVIIIa as compared to wild-type or naturallyoccurring factor Xa. In some embodiments, at least one amino acidsubstitution is present at amino acid position Arg306, Glu310, Arg347,Lys351, Lys414 or Arg424 in SEQ ID NOS. 3 and 7 (Arg125, Glu129, Arg165,Lys169, Lys230 or Arg240 in chymotrypsin numbering, respectively). Suchmodifications may be made to wild type fXa protein or to any of themodified fXa proteins or fragments described above.

In other embodiments, the antidote is a protein comprising the aminoacid sequence of a serine protease catalytic domain which can mimic theinhibitor binding capability of the fXa heavy chain. Such proteins mayinclude mammalian proteases such as plasma kallikrein, thrombin, trypsin(or its bacterial homolog subtilisin) which have been recombinantlymodified to lack serine protease activity capable of cleaving proteinsubstrates but still possess the structural characteristics of theactive site cleft.

Also provided by this invention are pharmaceutical compositionscontaining one or more of the modified factor Xa derivatives and apharmaceutically acceptable carrier. The compositions are administeredto a subject in need thereof in an amount that will provide the desiredbenefit, a reduction or stopping of bleeding. The compositions can beco-administered with any suitable agent or therapy that complements orenhances the activity of the factor Xa derivative. An example of such isa second agent capable of extending the plasma half-life of theantidote. Examples of suitable second agents include but are not limitedto an anti-fXa antibody recognizing the exosite of fXa heavy chain or analpha-2-macroglobulin bound fXa derivative. Formation of the complexbetween fXa derivative and a second agent (exosite antibody oralpha-2-macroglobulin) would block macromolecular interactions butretains the ability of active site dependent inhibitor bindings.Examples of anti-fXa antibodies suitable for co-administration includebut are not limited to those described in Yang Y. H., et al., J.Immunol. 2006, 1; 177(11):8219-25, Wilkens, M and Krishnaswamy, S., J.Bio. Chem., 2002, 277 (11), 9366-9374, and Church W R, et al., Blood,1988, 72(6), 1911-1921.

In some embodiments, a factor Xa protein is modified by chemical,enzymatic or recombinant means. For example, the active site Ser379 maybe chemically modified to dehydroalanine, and the Gla domain may beenzymatically removed by chymotrypsin digestion as described inExample 1. A modified fXa described herein may also be produced byrecombinant means by modifying the sequence of the cDNA encodingwild-type fX (SEQ ID NO. 2) described in more details in Example 7 fordirect expression of recombinant antidote (r-Antidote) or alternatively,a fX protein with the desired modification may be produced byrecombinant means followed by activation to the modified fXa by anactivator, such as a snake venom, e.g. Russell's viper venom, andcomplexes of fVIIa/tissue factor or fIXa/fVIIIa.

Subjects that will benefit from the administration of the compositionsdescribed herein and the accompanying methods include those that areexperiencing, or predisposed to a clinical major bleeding event or aclinically significant non-major bleeding event. Examples of clinicalmajor bleeding events are selected from the group consisting ofhemorrhage, bleeding into vital organs, bleeding requiring re-operationor a new therapeutic procedure, and a bleeding index of ≧2.0 with anassociated overt bleed. (Turpie A G G, et al., NEJM, 2001, 344:619-625.) Additionally, the subject may be experiencing or predisposedto a non-major bleeding event selected from the group consisting ofepistaxis that is persistent or recurrent and in substantial amount orwill not stop without intervention, rectal or urinary tract bleedingthat does not rise to a level requiring a therapeutic procedure,substantial hematomas at injection sites or elsewhere that arespontaneous or occur with trivial trauma, substantial blood loss morethan usually associated with a surgical procedure that does not requiredrainage, and bleeding requiring unplanned transfusion.

In some embodiments, the antidote is administered after theadministration of an overdose of a fXa inhibitor or prior to a scheduledelective surgery, which may expose subjects to the risk of hemorrhage.

In any of the methods described herein, it should be understood, even ifnot always explicitly stated, that an effective amount of the derivativeis administered to the subject. The amount can be empirically determinedby the treating physician and will vary with the age, gender, weight andhealth of the subject. Additional factors to be considered by thetreating physician include but are not limited to the identity and/oramount of factor Xa inhibitor, which may have been administered, themethod or mode that the antidote will be administered to the subject,the formulation of the antidote, and the therapeutic end point for thepatient. With these variables in mind, one of skill will administer atherapeutically effective amount to the subject to be treated. In stillanother aspect, the invention relates to a pharmaceutical compositionfor reversing or neutralizing the anticoagulant activity of a factor Xainhibitor administered to a subject, comprising administering aneffective amount of an antidote to the factor Xa inhibitor and apharmaceutically acceptable carrier, with the proviso that the antidoteis not plasma derived factor VIIa, recombinant factor VIIa, fresh frozenplasma, prothrombin complex concentrates and whole blood.

In some embodiments, the antidote is any one of the antidotes asdescribed above. In some embodiments, the antidote is conjugated with amoiety capable of extending the circulating half-life of the antidote.In some embodiments, the moiety is selected from the group consisting ofpolyethylene glycol, an acyl group, a liposome, a carrier protein, anartificial phospholipid membrane, and a nanoparticle. For example, anon-active site lysine or cysteine residue of a fXa derivative describedherein may be chemically modified to attach to a polyethylene glycolmolecule. Other methods provided in Werle, M. & Bernkop-Schnürch, A.Strategies to Improve Plasma Half Life Time of Peptide and ProteinDrugs, Amino Acids 2006, 30(4):351-367 may be used to extend the plasmahalf life of the antidotes of this invention.

In other embodiments of the invention, the half-life of the fXaderivative is improved by coupling the antidote to Fc carrier domains.In one embodiment, the antidote is coupled to an Fc fragment, such as animmunoglobulin peptide portion or an IgG1 fragment. In one embodiment, achimeric protein is contemplated which comprises the fXa derivative andthe immunoglobulin peptide portion. In yet another embodiment, the fXaderivative and the immunoglobulin peptide is coupled by a chemicalreaction, such as a disulfide bond with the human IgG heavy chain andkappa light chain constant regions.

In some embodiments, the pharmaceutical composition further comprises anagent capable of extending the plasma half-life of the antidote. Inanother aspect, the pharmaceutical composition has been co-formulatedwith an agent capable of extending the plasma half-life of the antidote.In some embodiments, the co-administered or co-formulated agent is ananti-fXa antibody recognizing the exosite of fXa or analpha-2-macroglobulin bound fXa derivative.

VI. Therapies

The present invention relates to a therapeutic method of preventing orreducing bleeding in a subject undergoing anticoagulant therapy. It iscontemplated that the antidotes or derivatives of the present inventionmay be short-duration drugs to be used in elective or emergencysituations which can safely and specifically neutralize a fXainhibitor's conventional anticoagulant properties without causingdeleterious hemodynamic side-effects or exacerbation of theproliferative vascular response to injury.

In one embodiment, the therapeutically effective amount of an antidoteexhibits a high therapeutic index. The therapeutic index is the doseratio between toxic and therapeutic effects which can be expressed asthe ratio between LD₅₀ and ED₅₀. The LD₅₀ is the dose lethal to 50% ofthe population and the ED₅₀ is the dose therapeutically effective in 50%of the population. The LD₅₀ and ED₅₀ are determined by standardpharmaceutical procedures in animal cell cultures or experimentalanimals. The antidotes or derivatives of this invention may beadministered once or several times when needed to neutralize the effectof a fXa inhibitor present in a subject's plasma. Preferably, theantidotes of this invention is sufficient when administered in a singledose.

It is contemplated that a typical dosage of the antidotes of theinvention will depend on the actual clinical setting and inhibitorconcentration in plasma. In in vitro assay, such as thrombin generation,clinical clotting assays such as aPTT, PT and ACT, a therapeuticallyeffective amount of an antidote is expected to produce a correction ofex vivo clotting activity of 10% or more. In vitro assays indicate thatan antidote/inhibitor ratio >1.0 should show reversal effect. Themaximum plasma concentration for antidote is expected to be in the micromolar range, probably between 10 micromolar or below.

In a clinical setting, one of the criteria in determining theeffectiveness of an antidote is that it produces any change of actualmeasures of bleeding. In clinical trials, categories of major bleedsinclude fatal hemorrhage, bleeds into vital organs (intracranial,intraocular, retroperitoneal, spinal, pericardial), any bleed requiringre-operation or a new therapeutic procedure (e.g., aspiration of anoperated knee, thoracotomy tube insertion for hemothorax, endoscopicelectrocoagulation, etc) or a bleeding index of ≧2.0 if it is associatedwith an overt bleed. The bleeding index is defined as the number ofunits of packed red cells or whole blood transfused plus the hemoglobinvalues before the bleeding episode minus the hemoglobin values after thebleed has stabilized (in grams per deciliter).

Another criterion for antidote efficacy in clinical settings is that itreduces clinically significant non-major bleeding. This category ofhemorrhages include bleeding that is not major but is more than usualand warrants clinical attention, including epistaxis that is persistentor recurrent and in substantial amount or will not stop withoutintervention; rectal or urinary tract bleeding that does not rise to alevel requiring a therapeutic procedure (e.g., new insertion of a Foleycatheter or cystoscopic inspection), substantial hematomas at injectionsites or elsewhere that are spontaneous or occur with trivial trauma;substantial blood loss; bleeding requiring unplanned transfusion. Asused herein, “substantial blood loss” refers to amount of blood lossthat is more than that amount usually associated with surgicalprocedure. Substantial blood loss leads to swelling that is managedconservatively because it falls short of requiring drainage.

In one embodiment, the derivatives of this invention have sufficientplasma circulating half life for substantially neutralizing the fXainhibitor present in plasma. Activated fXa has essentially nocirculating half life in humans, as it is effectively inhibited byATIII, TFPI and other plasma inhibitors (Fuchs, H. E. and Pizzo, S. V.,J. Clin. Invest., 1983, 72:2041-2049). Inactive fXa has been shown tohave a circulating half-life of 2-3 hours in humans. In a baboon model,the half-life of a fXa blocked in the active site by DEGR([5-(dimethylamino)1-naphthalenesulfonyl]-glutamylglycylarginylchloromethyl ketone) was approximately 10 hours or 2 hours, asdetermined by isotopic or enzyme-linked immunosorbent assays,respectively (Taylor, F. B. et al., Blood, 1991, 78(2):364-368).

It may be desirable to extend the half life of an antidote fXaderivative to 24-48 hours. It is contemplated that conjugation oraddition of one or more of the following moieties will increase theplasma half life of an antidote:

a) polyethylene glycol;b) an acyl group;c) liposomes and encapsulating agents;d) carrier proteins;e) artificial phospholipid membrane;f) immunoglobulin; andg) nanoparticle.The conjugation site may not be limited to special chain or residue solong as the conjugation does not mask the inhibitor binding site(s) ofthe antidote. The antidotes described herein may be administered incombination with any one or more than one of the compounds describedabove.

In general, administered antibodies have much longer half life thancirculating blood coagulation proteins. It is possible to use a complexconsisting of Gla-domain deficient fXa and an antibody bound to theexosite of fXa as an antidote with extended circulating half life.Formation of a complex between fXa and the antibody targeting theexosite may reduce interaction of an Gla-domain deficient fXa withmacromolecular substrates and inhibitors, such as prothrombin andantithrombin III, while leaving the active site cleft unperturbed sothat the complex can act as an antidote to bind active site directedsmall molecule inhibitor. Formation of α-2-macroglobulin-fXa complex canalso be of useful as an antidote for fXa small molecule inhibitors.

Efficacy of the antidotes in reversal of the anticoagulant activity offXa inhibitors as well as its procoagulant activity may be determined byin vitro assays and animal models by those of skill in the art. Examplesof in vitro assays are thrombin generation, clinical clotting assayssuch as aPTT, PT and ACT. An antidote of this invention is contemplatedto be capable of producing 10% or more correction of ex vivo clottingactivity. Several in vivo animal models of bleeding time and/or bloodloss in, for example, rodents, such as mice, dogs and primates, such asmonkeys, may be used to measure efficacy.

VII. Kits

The invention further provides kits or packages. In some embodiments,the kit of the present invention comprises: (a) a first containercontaining a fXa inhibitor for regular administration for the treatmentof thrombosis, and (b) a second container containing an antidote of thisinvention to be used in cases when there is an overdose of the fXainhibitor in (a) or when normal hemostasis needs to be restored to stopor prevent bleeding. In other embodiments, the kit further comprises alabel explaining when these two agents in (a) and (b) should be used.

The first and second container can be a bottle, jar, vial, flask,syringe, tube, bag, or any other container used in the manufacture,storage, or distribution of a pharmaceutical product. The package insertcan be a label, tag, marker, or the like, that recites informationrelating to the pharmaceutical composition of the kit. The informationrecited will usually be determined by the regulatory agency governingthe area in which the pharmaceutical composition is to be sold, such asthe United States Food and Drug Administration. Preferably, the packageinsert specifically recites the indications for which the pharmaceuticalcomposition has been approved. The package insert may be made of anymaterial on which a person can read information contained therein orthereon. Preferably, the package insert is a printable material, such aspaper, adhesive-backed paper cardboard, foil, or plastic, and the like,on which the desired information has been printed or applied.

Examples

The invention is further understood by reference to the followingexamples, which are intended to be purely exemplary of the invention.The present invention is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only. Any methods that are functionally equivalent arewithin the scope of the invention. Various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

Unless otherwise stated all temperatures are in degrees Celsius. Also,in these examples and elsewhere, abbreviations have the followingmeanings:

-   -   aa=amino acid    -   ab=antibody    -   ACT=activated clotting time    -   aPTT=activated partial thromboplastin time    -   CHO cell=Chinese hamster ovary cell    -   CHO dhfr(−)cells=CHO cells lacking dhfr gene    -   hr=hour    -   INR=international normalized ratio    -   IV=intravenous    -   kg=kilogram    -   M=molar    -   mg=milligram    -   mg/kg=milligram/kilogram    -   mg/mL=milligram/milliliter    -   min=minute    -   mL=milliliter    -   mM=millimolar    -   nm=nanometer    -   nM=nanomolar    -   PO=oral    -   PPP=platelet poor plasma    -   PRP=platelet rich plasma    -   PT=prothrombin time    -   RFU=relative fluorescence unit    -   s=second    -   TF=tissue factor    -   U/mL=units/milliliter    -   μL or uL=microliter    -   μM=micromolar    -   μg=microgram

Example 1 Preparation of des-Gla Anhydro-fXa by Chymotrypsin Digestion

Des-Gla anhydro-fXa was prepared according to the procedure of Morita,T. et al., J. Bio. Chem., 1986, 261(9):4015-4023 by incubatinganhydro-fXa, in which dehydroalanine replaces the active-site serine,with chymotrypsin in 0.05 M Tris-HCl, 0.1 M NaCl, at pH 7.5 and 22° C.for 60 minutes. In a typical experiment setting, 0.5milligrams/milliliter (mg/mL) anhydro-fXa was incubated with 5units/milliliter (U/mL) α-chymotrypsin-agarose beads with gentleagitation. At the end of the reaction, the α-chymotrypsin-agarose beadswere removed by centrifugation or filtration. This was followed byincubation with excess amount of inhibitors4-amidino-phenyl-methane-sulfonyl fluoride (APMSF), tosyl-L-lysinechloromethyl ketone (TLCK), and tosyl-L-phenylalanine chloromethylketone (TPCK) to quench the residual fXa activity or any activity ofchymotrypsin possibly leached from the beads. Gla-domain fragment andinhibitors were removed from the final product, des-Gla anhydro-fXa, byan Amicon Ultra Centrifugal filter device (YM10 membrane) or byconventional dialysis. Concentrating or buffer exchange, if necessary,was also achieved at the same time. The Gla-domain containinganhydro-fXa was prepared according to the procedure reported by Nogami,et al., J. Biol. Chem. 1999, 274(43):31000-7.

The Gla-containing anhydro-fXa was prepared according to the procedurereported by Nogami et al., J. Biol. Chem., 1999, 274(43):31000-7. Asshown in FIG. 5, the Gla-containing anhydro-fXa has diminished enzymaticactivity but is capable of binding fXa inhibitors such as betrixaban.This is described in detail in Example 4.

α-Chymotrypsin-agarose bead was purchased from Sigma and the specificactivity (U/mL) was based on manufacturer's data for the specific lotnumber used.

Chymotrypsin digestion of active fXa can be carried out according toabove procedure without using APMSF. Clotting activity of active fXa wasdetermined before the chymotrypsin digestion, and after 15, 30 and 60minutes of chymotrypsin digestion according to the procedure describedin Example 3 below. FIG. 7 shows complete loss of clotting activityafter 30 minutes of chymotrypsin digestion. The incubation time wereextended to 60 minutes to ensure complete removal of the Gla domain.

Example 2 Thrombin Generation Assay in Platelet Poor Plasma (PPP) orPlatelet Rich Plasma (PRP)

In this example, human platelet poor or platelet rich plasma sampleswere prepared from blood of healthy donors drawn into 0.32% citrate. PRPand PPP were prepared by spinning the anticoagulated blood at −100×gravity or 1000× gravity for 20 minutes, respectively, at roomtemperature. 75-100 microliter (uL) plasma was mixed with CaCl₂ andZ-Gly-Gly-Arg-aminomethylcoumarin (Z-GGR-AMC, a thrombin fluorogenicsubstrate). Tissue factor (Innovin, Dade Behring) was added to initiatethe generation of thrombin. For a typical experiment, the reactionmixture contained 15 millimolar (mM) Ca²⁺, 100 micromolar (μM)Z-GGR-AMC, and 0.1 nanomolar (nM) tissue factor (TF) (Innovin). Thrombinformation was monitored continuously at 37° C. by a fluorometric platereader (Molecular Devices) measuring the relative fluorescence units(RFU). Inhibitor and antidote, when present, were pre-incubated withplasma for 20 minutes at room temperature before initiation of thrombingeneration.

The results of various experiments using this assay may be found inFIGS. 4, 6, and 9.

Example 3 Clotting Prolongation Assays

Two clotting assay formats were used to test the effects of factor Xainhibitors and the antidote on clotting prolongation. In the firstformat, a 96-well plate was used to measure multiple samples at the sametime. In the second assay format, aPTT was measured with a conventionalcoagulation instrument (MLA Electra 800 automatic coagulation timer).

In the 96-well plate format method, human platelet poor plasma orplatelet rich plasma was prepared similarly as procedures in Example 2.75-100 μL plasma was recalcified with CaCl₂, incubated at 37° C. for 3minutes and clot formation was initiated by adding tissue factor(Innovin, Dade Behring) or an aPTT reagent (Actin FS, Dade Behring).Change of OD405 was monitored continuously by a plate reader (MolecularDevices). Clotting time was defined as the time (second) when the halfmaximal value of absorbance (OD405 nm) change was reached. Factor Xainhibitor and antidote, when present, were pre-incubated with plasma atroom temperature for 20 minutes before initiation of the reaction.

When an active fXa was tested for its clotting activity as shown in FIG.7, 75-100 uL fX deficient plasma (George King Bio-Medical, Inc.) wasrecalcified with CaCl₂, incubated at 37° C. for 3 minutes and fXaproducts following chymotrypsin digestion was added to the plasma toinitiate clot formation. Change of OD405 was continuously monitored by aplate reader as described before.

In FIG. 13, the effect of 400 nM betrixaban on aPTT prolongation ofnormal human plasma and the reversal of betrixaban inhibitory effect byantidote des-Gla anhydro-fXa was measured with a MLA Electra 800Automatic coagulation timer. 100 μL pooled human plasma was mixed with400 nM betrixaban and different concentration of antidote. aPTT reagent(Actin FS, Dade Behring) and CaCl₂ were added per manufacturer'sinstructions for measurement of clotting times.

Results of additional experiments using this assay may be found in FIGS.10 and 11.

Example 4 Reversal of Inhibition of fXa by Betrixaban by Anhydro-fXa ordes-Gla Anhydro-fXa

To measure the inhibition of fXa activity by betrixaban and reversal ofits inhibitory effect, purified active fXa, different concentrations ofbetrixaban and anhydro-fXa or des-Gla anhydro-fXa were added to 20 mMTris, 150 mM NaCl, 5 mM Ca²⁺, and 0.1% Bovine Serum Albumin (BSA). Afterincubation at room temperature for 20 minutes, 100 μM Spectrozyme-fXa (afactor Xa chromogenic substrate, Chromogenix) was added to the mixtureand the rate of substrate cleavage was monitored continuously for 5minutes at 405 nanometer (nm) by a plate reader. In FIG. 5, thechromogenic activity was normalized to active fXa in the absence of anyinhibitor. Initial velocity of product formation as a function ofinhibitor and antidote concentration was analyzed by nonlinearregression to estimate the affinity of betrixaban to the antidote (FIG.8).

The effect of the antidote des-Gla anhydro-fXa on thrombin activitytoward a chromogenic substrate S2288 (200 μM) was measured similarly asbefore with or without Argatroban, a specific small molecule IIainhibitor. As expected, the antidote (538 nM) does not affect theamidolytic activity of IIa (5 nM) or its inhibition by 50 nM Argatroban.

Example 5 Preparation of fXa with Decarboxylated γ-Carboxyglutamic AcidResidues

A fXa derivative with decarboxylated γ-carboxyglutamic acid residues canbe prepared by treating fXa protein, for example, based on the procedurereported by Bajaj, et al. J. Biol. Chem., 1982, 257(7):3726-3731. 2 to 5mg of purified or recombinant fXa in 2 mL of 0.1 Molar ammoniumbicarbonate at pH 8.0 is lyophilized. The resulting powder is sealedunder a vacuum of less than 20 μm and heated at 110° C. for variousperiods of time to obtain decarboxylated fXa.

Example 6 Preparation of Recombinant des-Gla fXa-S379A

The fXa derivatives may be produced by recombinant DNA method with oneof the following procedures based on fX cDNA (SEQ ID NO. 2) forexpressing fX (SEQ ID NOS. 1, 3) or fXa derivatives (SEQ ID NOS. 4, 5,9, and 11) in a suitable host organism according to general proceduresof mutagenesis and molecular biology.

Recombinant fX and fX derivatives can be expressed in, for example,human embryonic kidney cells HEK293 based on procedures described inLarson, P. J., et al., Biochem., 1998, 37:5029-5038, and Camire, R. M.,et al., Biochem., 2000, 39, 14322-14329. Recombinant fX can be activatedto rfXa by factor X activator Russell's Viper Venom (RVV). rfXa can befurther processed to des-Gla anhydro-fXa based on procedures describedin Example 1.

Recombinant fX-S379A (5195A in chymotrypsin numbering) with the activesite serine residue being replaced by alanine, and preferably theactivated fXa mutant, rfXa-S379A, may be expressed, for example, inChinese Hamster Ovary (CHO) cells based on procedures described by Sinhaet al., Protein Expression and Pur 1992, 3: 518-524; Wolf, D. L. et al.,J. Biol. Chem., 1991, 266(21):13726-13730.

Des-Gla fXa-S379A may be prepared by chymotrypsin digestion of fXa-S379Aaccording to procedures described in Example 1.

More preferably, Des-Gla fXa-S379A may be expressed directly accordingto previous procedures with deletion of Gla-domain fragment bymutagenesis procedures. For example, recombinant protein expression canbe used to express: des-Gla(1-39)-fXa-S379A, after removal of Gla-domainfragment 1-39 of SEQ ID NO. 3; des-Gla(1-44)-fXa-S379A, equivalent toSEQ ID NO. 10 with dehydro-alanine being replaced by alanine; anddes-Gla(1-45)-fXa-S379A with entire Gla-domain being removed (SEQ ID NO.11).

Further truncations at EGF1 or EGF1 plus EGF2 domain (FIG. 2) can alsobe made to express des(1-84)-fXa-S379A or des(1-128)-fXa-S379Aderivatives.

Example 7 Expression of Recombinant fXa Mutant in CHO Cell

This example describes the recombinant protein expression construct andthe cell line for the direct expression of a Gla-domainless fXa-S379A(S195A in chymotrypsin numbering) variant. The recombinant antidote doesnot require activation or chemical modification steps necessary toproduce the pd-Antidote and has comparable affinity to the plasmaderived protein in the in vitro assays discussed herein.

In this example, a fXa mutant (SEQ ID NO. 13, Table 25) was directlyexpressed in CHO cell (see FIG. 14 for expression vector) and functionalprotein was purified from conditioned medium as described below.Recombinant antidote (r-Antidote) functional activity was tested invitro and in animal model (Example 8).

PCR was used to mutate the cDNA sequence of fX (SEQ ID NO. 2) in threeregions. The first mutation was the deletion of 6-39 aa in theGla-domain of FX (SEQ ID NO. 3, FIG. 3). The second mutation wasreplacing the activation peptide sequence 143-194 aa with RKR (SEQ IDNO. 16). This produced a RKRRKR (SEQ ID NO. 17) linker connecting thelight chain and the heavy chain. Upon secretion, this linker is removedin CHO resulting in a two-chain fXa molecule. The third mutation ismutation of active site residue S379 to an Ala residue.

The polypeptide produced by the cDNA (SEQ ID NO. 16) just described isdescribed in Table 24 (SEQ ID NO. 12). The alignment of the cDNA to thepolypeptide is shown in Table 29. The two-chain fXa molecule producedafter secretion is a light chain fragment described in Table 26 (SEQ IDNO. 14) and a heavy chain fragment described in Table 27 (SEQ ID NO.15).

The first 1-5 aa in fX sequence was reserved and used to connect thepolypeptide of fXa mutant to the prepro peptide of fX (SEQ ID NO. 1,FIG. 1), ensuring proper processing of the prepro peptide in fXa mutant.

DNA sequence encoding the polypeptide of fXa mutant described above wassequenced and inserted to the expression vector shown in FIG. 14. Thepolynucleotide of the expression vector is shown in SEQ ID NO. 18.Plasmid DNA was linearized and transfected into CHO dhfr(−) cells. Cellswere selected using tetrahydrofolate (HT) deficient media plusmethotrexate (MTX). Stable clones were screened for high proteinexpression using a fX ELISA kit (Enzyme Research Laboratories, CatalogueNumber FX-EIA). FXa mutant protein was expressed in serum free mediumand conditioned medium was harvested and processed for purification.

Target protein in the conditioned medium can be isolated by ion exchangechromatography and subsequently purified by single step affinitychromatography (such as an anti-fXa antibody coupled to a matrix) or bya combination of several chromatography steps such as hydrophobic andsize exclusion matrices. The affinity purifications may includechromatographic material that selectively binds to fXa active sitecleft, such as benzamidine-sepharose or soybean trypsininhibitor-agarose (STI-Agarose).

FIG. 15A shows the Western blots of affinity (STI-Agarose, Sigma Catalog#T0637) purified fXa mutant using monoclonal antibodies (Enzyme ResearchLaboratories, FX-EIA) recognizing fX heavy and light chain,respectively. Upon reduction of the disulfide bond which connects thelight and heavy chain, r-Antidote shows the heavy chain band of expectedmobility (similar to plasma derived fXa) in the Western blot. Deletionof amino acid residues (numbered 6 through 39) in the Gla-domain of fXamutant results in a lower molecular weight band for the light chain ofr-Antidote compared to plasma derived fXa. Position of molecular weightmarkers can also be seen on the blot. FIGS. 15B and 15C show a SDS-PAGEand Western blot of purified r-Antidote by ion exchange and affinitypurification followed by size exclusion chromatography using a Superdex75 10/300 GL column (GE Healthcare, Cat #17-5174-01).

Example 8 In Vivo Mouse Model

The pharmacokinetic and pharmacodynamic (PK-PD) profile of betrixaban inmale C57Bl/6 mice with or without administrating antidote were tested.Single oral administration of betrixaban was dosed at 0, 15, 25, and 75mg/kg for controls groups. 15 mg/kg was used for antidote treated group.A single intravenous (IV) injection of antidote (300 ug/200 μL) orvehicle (normal saline, 200 μL) was administered 5 minutes prior to the1.5 hr. time point.

At 1.5, 2.0, and 4.0 hrs following oral administration of betrixaban,mice were anesthetized with a ketamine cocktail (SC) and exsanguinatedvia cardiac puncture. Blood samples (0.5 mL) were obtained in 50 μL,trisodium citrate. Whole blood INR was measured using Hemochron Jr.cartridges (International Technidyne Corporation) per the manufacture'sinstructions. Mouse platelet poor plasma was prepared by centrifugationfor betrixaban and antidote (ELISA) plasma concentration determinations.

For recombinant antidote (r-Antidote) experiment, mice were orally dosedwith betrixaban at 0, 15, 25, and 75 mg/kg for control groups. 15 mg/kgwas used for antidote (300 μg/200 μL) treated group. Samples were takenat 1.5 hr after oral administration of betrixaban (5 min. followingantidote injection).

As shown in FIGS. 16 and 17A-B and Tables 1 and 2, single injection (300μg, IV) of plasma derived antidote (pd-Antidote) or recombinant fXamutant (r-Antidote) to mice following administration of betrixaban (15mg/kg, PO) effectively captured the inhibitor in vivo. PK-PD correlationof whole blood INR and antidote plasma concentration (Tables 1 and 2)indicated >50% reduction of functional betrixaban based on INRmeasurements, and justified effective neutralization of fXa inhibitorsby the antidote via multiple injections or other regimes. It iscontemplated that these results demonstrate that the fXa derivatives ofthis invention have potential of acting as universal antidotes toreverse the anticoagulant effect of fXa inhibitors in patients withbleeding or other medical emergencies.

TABLE 1 PK-PD correlation in pd-Antidote treated mice at 1.5 hr after 15mg/kg Betrixaban oral administration (5 min after antidote injection)pd-Antidote treated animal 1 2 3 4 5 6 7 Mean Betrixaban 673 793 1170415 217 664 879 687 (ng/mL) Expected 4.2 4.5 5.2 3.3 2.3 4.1 4.7 4.0 INRMeasured 2.3 2.3 3.3 0.8 0.8 1.5 2.0 1.9 INR % Correction 63.9 66.6 52.3100 100 83.1 74.4 77.2

TABLE 2 PK-PD correlation in r-Antidote treated mice at 1.5 hr after 15mg/kg Betrixaban oral administration (5 min after antidote injection)r-Antidote treated animal 1 2 3 4 Mean Betrixaban (ng/mL) 434 262 335494 381 Expected INR 3.2 2.5 2.8 3.5 3.0 Measured INR 2.0 0.9 1.2 0.91.3 % Correction 50.0 94.1 80.0 93.6 77.3

FIGS. 22A-B show mouse experiment with a single IV injection (1injection) or two injections (2 injections) of the r-antidote (n=5 pergroup, 312 ug/200 ul r-Antidote) following oral administration ofbetrixaban (15 mg/kg). For the single injection group, mouse bloodsamples were taken at 1 hr. following oral administration of betrixaban.Vehicle (control_(—)1) or r-Antidote (1 injection) was administered 5min prior to the 1 hr. time point. For the double injection group,vehicle or r-Antidote was injected at 55 min and repeated at 115 minfollowing oral administration of betrixaban. Mouse blood samples weretaken at 2 hr. for vehicle (control_(—)2) and r-Antidote (2 injections)treated mice. Measured INR as a function of antidote/betrixaban ratio inmouse plasma following single or double injections of the antidote wasshown in FIG. 22 B.

Example 9 In Vitro Reversal of Rivaroxaban and Apixaban by Antidote

As expected, the antidotes contemplated by this invention were also ableto bind and neutralize other active site directed fXa inhibitors. Tables3 and 4 show in vitro correction of inhibition by betrixaban,rivaroxaban and apixaban by pd-Antidote and r-Antidote. Purified fXa(3.0 nM), inhibitor (7.5 nM), and different concentrations of antidotewere incubated for 10 min at 22° C. in a buffer with 20 mM Tris, 150 mMNaCl, 0.1% BSA, pH7.4. fXa activity was assayed similar to Example 4.

TABLE 3 % Correction of inhibition by fXa inhibitors pd-Antidote (nM)Betrixaban Rivaroxaban Apixaban 0 0 0 0 10.2 13.1 10.6 6.5 20.4 34.837.4 11.4 40.7 47.1 46.8 15.0 61.1 68.4 55.7 40.3 101.8 67.5 69.4 52.3162.9 80.5 74.0 56.0 203.7 82.6 72.6 60.2

TABLE 4 % Correction of inhibition by fXa inhibitors r-Antidote (nM)Betrixaban Rivaroxaban Apixaban 0 0 0 0 9.3 21.5 23.2 13.3 18.6 52.754.2 33.5 37.2 75.5 72.6 49.9 55.8 86.5 79.9 59.2 93.1 94.9 89.1 64.4148.9 99.3 96.7 74.8 186.1 99.5 94.8 72.6

As shown in Table 3, 204 nM pd-Antidote produces at least 60% correctionof the inhibitory effects of tested inhibitors, while in Table 4 >95%correction of inhibition was achieved by the r-Antidote (186 nM) forbetrixaban and rivaroxaban, and >70% reversal of apixaban.

Example 10 In Vitro Reversal of Betrixaban by r-Antidote

In Table 5, the effect of recombinant antidote protein on reversal ofanticoagulation by betrixaban was tested in a human plasma clottingassay. The effect of 300 nM and 400 nM betrixaban on aPTT prolongationof plasma and the reversal of inhibitory effect was measured by a MLAElectra 800 Automatic coagulation timer. 100 μL pooled citrateanticoagulated human plasma was mixed with 300 nM or 400 nM betrixabanand different concentrations of antidote. aPTT reagent (Actin FS, DadeBehring) and CaCl₂ were added per manufacturer's instructions.

TABLE 5 r-Antidote reversal of anticoagulant activity of betrixaban aPTTFold % Correction of (sec) Change anticoagulation Control human plasma35.2 1.00 — 300 nM Betrixaban 61.8 1.76 — 300 nM Betrixaban + 38.3 1.0988 570 nM r-Antidote 300 nM Betrixaban + 38.2 1.09 88 760 nM r-Antidote300 nM Betrixaban + 38.1 1.08 90 1140 nM r-Antidote 400 nM Betrixaban66.3 1.88 — 400 nM Betrixaban + 47.1 1.34 61 380 nM rAntidote 400 nMBetrixaban + 39.9 1.13 85 570 nM rAntidote 400 nM Betrixaban + 39.9 1.1385 760 nM rAntidote 400 nM Betrixaban + 37.8 1.07 92 1140 nM rAntidote400 nM Betrixaban + 39.4 1.12 86 1520 nM rAntidote 1140 nM rAntidote38.9 1.11 — 1520 nM rAntidote 38.8 1.10 —

Example 11 In Vitro Reversal of Low Molecular Weight Heparin (“LMWH”) byr-Antidote

In FIG. 18, the effect of r-Antidote to reverse the inhibitory effect ofLMWH enoxaparin (Sanofi-Aventis) was tested by turbidity changes inhuman plasma. Enoxaparin (0-1.25 U/mL) was incubated at 22° C. for 20min with or without 508 nM r-Antidote. Turbidity changes were measuredaccording to procedures described in Example 3. 508 nM r-Antidotesubstantially corrected (>75%) the inhibitory effect of 0.3125-1.25 U/mLEnoxaparin.

In FIG. 19, the effect of r-Antidote on reversal of anticoagulation by alow molecular weight heparin (LMWH enoxaparin, Sanofi-Aventis) wastested in a human plasma clotting assay. The effect of 1 antiXa Unit/mLLMWH on aPTT prolongation of plasma and the reversal of inhibitoryeffect was measured by a MLA Electra 800 Automatic coagulation timer.100 μL pooled citrate anticoagulated human plasma was mixed withenoxaparin and different concentrations of antidote. Prior tomeasurement of clotting time, aPTT reagent (Actin FS, Dade Behring) andCaCl₂ were added per manufacturer's instructions. Addition of 1.14 μMrecombinant antidote produced a 52% correction of anticoagulationproduced by 1 Unit/mL enoxaparin.

Example 12 In Vitro Reversal of Rivaroxaban by r-Antidote

In FIG. 20, the effect of recombinant antidote protein on reversal ofanticoagulation by a small molecule factor Xa inhibitor (rivaroxaban,Bay 59-7939) was tested in a human plasma clotting assay. As reported byPerzborn et al., J. Thromb. Haemost. 3:514-521, 2005; prothrombin timemeasurements are an accurate method for evaluating the anticoagulanteffect of rivaroxaban. The effect of 1 μM rivaroxaban on prothrombintime (PT) prolongation of pooled human plasma and the reversal ofinhibitory effect was measured by a MLA Electra 800 Automaticcoagulation timer. 100 μL pooled citrate anticoagulated human plasma wasmixed with rivaroxaban and different concentrations of antidote. Priorto measurement of clotting time, rabbit brain Thromboplastin C Plusreagent (Dade Behring) was added to plasma samples per manufacturer'sinstructions. Addition of 1.9 μM recombinant antidote produced a 100%correction of anticoagulation produced by 1 μM rivaroxaban.

Example 13 In Vitro Reversal of Apixaban by r-Antidote

In Table 6, the effect of recombinant antidote protein on reversal ofanticoagulation by apixaban was tested in a human plasma clotting assay.As reported by Pinto et al., J. Med. Chem. 55(22):5339-5356, 2007;prothrombin time (PT) measurements are an accurate method of evaluatingthe ex vivo anticoagulant effects of apixaban. The effect of 1 μM and1.5 μM apixaban on prothrombin time (PT) prolongation of pooled humanplasma and the reversal of inhibitory effect was measured by a MLAElectra 800 Automatic coagulation timer. 100 μL pooled citrateanticoagulated human plasma was mixed with apixaban and differentconcentrations of antidote. Prior to measurement of clotting time,rabbit brain Thromboplastin C Plus reagent (Dade Behring) was added toplasma samples per manufacturer's instructions. Addition of 1.9 μMrecombinant antidote produced a 97% correction of anticoagulationproduced by 1.5 μM apixaban.

TABLE 6 r-Antidote reversal of anticoagulant activity of Apixaban PTFold (sec) Change Control human plasma 14.1 — 1 μM apixaban 16.4 1.16 1μM apixaban + 15.3 1.09 380 nM rAntidote 1 μM apixaban + 14.9 1.06 760nM rAntidote 1 μM apixaban + 14.2 1.01 1.14 μM rAntidote 1 μM apixaban +14.2 1.01 1.52 μM rAntidote 1.5 μM apixaban 18.4 1.31 1.5 μM apixaban +14.6 1.04 1.52 μM rAntidote 1.5 μM apixaban + 14.3 1.01 1.90 μMrAntidote 1.52 μM rAntidote 14 — 1.90 μM rAntidote 14.2 —

Example 14 In Vitro Inhibition of Argatroban by des-Gla Anhydro-fXa

To measure the inhibition of thrombin activity by argatroban andreversal of its inhibitory effect, purified human thrombin (5 nM),argatroban (50 nM) and different concentrations of antidote des-Glaanhydro fXa were added to a buffer containing 20 mM Tris, 0.15 M NaCl, 5mM Calcium chloride, 0.1% bovine serum albumin, pH 7.4. After incubationat room temperature for 20 min, an amidolytic substrate S2288 (200 uM)was added to the mixture and the rate of p-nitroanilide substratecleavage was monitored by absorbance at 405 nm. The results arepresented in FIG. 12.

Example 15 Reversal of Activity of Direct fXa Inhibitors by r-Antidote

To measure the inhibition of fXa activity by a small molecule fXainhibitor and reversal of its inhibitory effect, purified active humanplasma derived fXa (3 nM), different concentrations of inhibitor (0,2.5, 5.0, 7.5 nM) and r-Antidote (0-125 nM) were added to a buffercontaining 20 mM Tris, 150 mM NaCl, 5 mM Ca²⁺, and 0.1% Bovine SerumAlbumin (BSA) in a 96-well plate. After incubation at room temperaturefor 20 minutes, 100 μM Spectrozyme-fXa (a factor Xa chromogenicsubstrate, American Diagnostica) was added to the mixture and the rateof substrate cleavage was monitored continuously for 5 minutes at 405nanometer (nm) by a plate reader (Molecular Devices). The test wascarried out in a total volume of 200 Initial velocity of substratecleavage as a function of inhibitor and antidote concentration wasanalyzed by nonlinear regression to estimate the affinity of theantidote for the inhibitors. Dynafit software was used for dataanalysis.

FIGS. 23A-C shows r-Antidote reversal of the inhibitory effect on fXaactivity by: Rivaroxaban (A), Betrixaban (B) and Apixaban (C). The curvefits were drawn using estimated affinity of r-Antidote (Kd) and reportedKi of human plasma fXa for each inhibitor as shown in Table 7. Theinhibition constants (Ki) are reported in the following literaturereferences: Rivaroxaban (Perzborn E, Strassburger J, Wilmen A, PohlmannJ, Roehrig S, Schlemmer K H, Straub A. J Thromb Haemost. 2005 March;3(3):514-21), Betrixaban (Sinha U, Edwards S T, Wong P W, et al.Antithrombotic activity of PRT54021, a potent oral direct factor Xainhibitor, can be monitored using a novel prothrombinase inhibitionbioassay. Blood 2006; 108: Abstract 907), and Apixaban (Pinto D J, OrwatM J, Koch S, Rossi K A, Alexander R S, Smallwood A, Wong P C, Rendina AR, Luettgen J M, Knabb R M, He K, Xin B, Wexler R R, Lam P Y. J MedChem. 2007 Nov. 1; 50(22):5339-56. Epub 2007 Oct. 3).

The results shown in FIGS. 23A-C and Table 7 indicate that r-Antidotecan bind these direct fXa inhibitors with high affinity and is able todose-dependently reverse their inhibitory effects on human fXa.

TABLE 7 Estimated affinity of r-Antidote for small molecule fXainhibitors Inhibitor Ki, Xa (nM)* Kd, r-antidote (nM) Rivaroxaban 0.43.3 Betrixaban 0.1 0.7 Apixaban 0.1 1.1 *Inhibition constants reportedin the literature

Example 16 Reversal of Ex Vivo Anticoagulant Activity of Direct fXaInhibitors by r-Antidote

Rivaroxaban (Xarelto™, Bay 59-7939) is a direct fXa inhibitor indicatedfor prevention of venous thromboembolism in patients undergoingorthopedic surgery. As reported by Perzborn et al., J. Thromb. Haemost.3:514-521, 2005; prothrombin time (PT) measurements are an accuratemethod for evaluating the anticoagulant effect of rivaroxaban.Clinically effective doses of rivaroxaban produce peak plasmaconcentrations as high as 318 ng/ml (730 nM, Kubitza et al, Eur. J.Clin. Pharmacol. 61:873-880, 2005). In order to mimic the anticoagulanteffect of supratherapeutic concentrations, at levels likely to beimplicated in clinically significant bleeding scenarios, the feasibilitywas tested of reversing concentrations of rivaroxaban which were higherthan 730 nM.

The effect of 1 μM rivaroxaban on prothrombin time (PT) prolongation ofpooled human plasma (prepared as reported in Sinha U, Lin P H, Edwards ST, Wong P W, Zhu B, Scarborough R M, Su T, Jia Z J, Song Y, Zhang P,Clizbe L, Park G, Reed A, Hollenbach S J, Malinowski J, Arfsten A E.Arterioscler Thromb Vasc Biol. 2003 Jun. 1; 23(6):1098-104. Epub 2003May 15) was measured in a MLA Electra 800 Automatic coagulation timer.Combination of citrate anticoagulated plasma from eight healthyvolunteer donors was used for the experiments. In order to measureclotting time, rabbit brain Thromboplastin C Plus reagent (Dade Behring)was added to plasma samples (100 uL) per manufacturer's instructions.

As shown in FIG. 24, baseline PT (14.1 sec) was prolonged to 23.4seconds upon addition of 1 μM rivaroxaban. The anticoagulant effect ofrivaroxaban was dose-dependently and completely reversed by addition ofr-Antidote whereas addition of 1.9 μM r-Antidote alone did not produce anoticeable effect on PT (14.2 sec).

Apixaban (BMS-562247) is a direct fXa inhibitor being tested forprevention of thromboembolic events in atrial fibrillation patients andfor prevention of venous thromboembolism in patients undergoingorthopedic surgery (Lassen M R, Davidson B L, Gallus A, Pineo G, AnsellJ, Deitchman D. J Thromb Haemost. 2007 December; 5(12):2368-75. Epub2007 Sep. 15. As reported by Luettgen et al., Blood 108 (11) Abstract4130, 2006, PT measurements are an accurate method for evaluating theanticoagulant effect of apixaban.

FIG. 25 shows prolongation of PT by apixaban and reversal of its effectsby addition of r-Antidote.

Example 17 Reversal of Inhibition of Indirect fXa Inhibitors byr-Antidote

A turbidity assay was used to test the effect of fXa inhibitors andr-Antidote on prolongation of clotting times. In this format, a 96-wellplate was used to measure multiple samples at the same time. Humanplatelet poor plasma was prepared as in Example 2. 75-100 μL plasma wasrecalcified with CaCl₂, incubated at 37° C. for 3 minutes and clotformation was initiated by adding tissue factor (Innovin, Dade Behring)or an activated partial thromboplastin time reagent (aPTT, Actin FS,Dade Behring). Change of OD405 was monitored continuously by a platereader (Molecular Devices). Clotting time was defined as the time(seconds) for reaching the half maximal value for change in absorbance(OD405 nm). FXa inhibitor (low molecular weight heparin such asenoxaparin, Aventis Pharma) and r-Antidote, when present, werepre-incubated with plasma at room temperature for 20 minutes beforeinitiation of the reaction.

FIG. 26 shows change of clotting parameter (fold) when 1 U/ml lowmolecular heparin (enoxaparin, Lovenox) was added to human platelet poorplasma, followed by addition of different amounts of r-Antidote. Whenturbidity change was measured following addition of aPTT reagent, 1 U/mlenoxaparin produced greater than 5-fold extension of clotting timecompared to control plasma. The prescribed dose (1 mg/kg subcutaneousdosing) of enoxaparin in acute coronary syndrome patients corresponds toapproximately 1 U/mL. This pharmacodynamic marker (anti-fXa unit) hasbeen specifically developed for LMWHs and correlated with clinicalefficacy and safety (Montalescot G, Collet J P, Tanguy M L, Ankri A,Payot L, Dumaine R, Choussat R, Beygui F, Gallois V, Thomas D.Circulation. 2004 Jul. 27; 110(4):392-8. Epub 2004 Jul. 12). In order tomimic the anticoagulant effect that is likely to be implicated when apatient's anticoagulation status needs to be reversed due to emergencysurgery, we tested the feasibility of reversing therapeuticconcentrations of enoxaparin. As shown in FIG. 26, r-Antidotedose-dependently reversed the anticoagulant effect of enoxaparin (1 fXaUnit/ml) with near complete correction of aPTT being attained uponaddition of 4 uM r-Antidote.

Example 18 Reversal of Rivaroxaban Induced Anticoagulation byIntravenous Administration of r-Antidote in Rats

Rats were anesthetized with intraperitoneal administration of ketaminecocktail, rapidly catheterized for jugular vein administration ofrivaroxaban and antidote and a second catheter was placed for serialblood sampling from femoral vein. Blood sampling catheter patency wasmaintained by slow infusion of normal saline between samples. Rats wereadministered rivaroxaban at 0.25 mg/kg/hr IV or vehicle (50%polyethyleneglycol in water) for 30 minutes (5.24 mL/kg/hr). At 30minutes, the rivaroxaban infusion was discontinued and r-Antidoteadministered at 1.0 or 3.4 mg as an IV (2 ml) bolus over 5 minutes.Serial blood samples anticoagulated with 3.2% sodium citrate (1:10dilution) were obtained for measurement of whole blood INR, rivaroxabanplasma concentrations and antidote concentrations at 0, 30 (before theend of rivaroxaban infusion), 35 (end of bolus treatmentadministration), 60, 90 and 120 minutes. Whole blood INR and PTmeasurements were determined on a Hemochron Jr using the citrated PTcartridges. Whole blood INR and PT are used for monitoring patientsunder warfarin anticoagulation and are reported as validated methods forevaluating extent of anticoagulation. Plasma samples were analyzed forrivaroxaban concentration using high-performance-liquid-chromatographywith tandem mass spectrometry. Quantitation was performed using acalibration standard curve generated from weighted least squareregression analysis. r-Antidote concentrations were determined by ELISAas described in Example 23. FIG. 27 shows dose responsive reversal ofthe effect of rivaroxaban following administration of r-Antidote. Theanticoagulation status of dosed rats was quantitated by a point of careclotting assay (Whole Blood INR). The difference in whole blood INRbetween rivaroxaban treated and r-Antidote dosed groups werestatistically significant (p≦0.004 for 1 mg dose and p≦0.001 for 3.4 mgdose) by Student's T test (unpaired two tailed).

Example 19 Measurement of Reduction of Unbound Plasma Concentration ofRivaroxaban Upon Dosing of r-Antidote

Rats were anesthetized with intraperitoneal administration of ketaminecocktail, rapidly catheterized for jugular vein administration ofrivaroxaban and antidote and a second catheter was placed for serialblood sampling from femoral vein. Blood sampling catheter patency wasmaintained by slow infusion of normal saline between samples. Rats wereadministered rivaroxaban at 0.25 mg/kg/hr IV or vehicle (50%polyethyleneglycol in water) for 30 minutes (5.24 mL/kg/hr). At 30minutes, the rivaroxaban infusion was discontinued and r-Antidoteadministered at 1.0 or 3.4 mg as an IV (2 ml) bolus over 5 minutes.Serial blood samples anticoagulated with 3.2% sodium citrate (1:10dilution) were obtained for measurement of whole blood INR, rivaroxabanplasma concentrations (total and unbound concentrations) and antidoteconcentrations at 0, 30 (before the end of rivaroxaban infusion), 35(end of bolus treatment administration), 60, 90 and 120 minutes.Fraction of rivaroxaban not bound to rat plasma proteins and/or tor-antidote protein was determined by an ultrafiltration method usingMicrocon devices. Plasma samples were analyzed for rivaroxabanconcentration using high-performance-liquid-chromatography with tandemmass spectrometry. Quantitation was performed using a calibrationstandard curve generated from weighted least square regression analysis.Results are shown in FIG. 28.

Example 20 Sustained Reversal of Rivaroxaban Activity Followingr-Antidote Dosing in Rats

Rats were anesthetized and catheterized as described in example 18. Ratswere administered rivaroxaban or vehicle at 0.25 mg/kg/hr by intravenousadministration (vehicle=50% polyethylene glycol in water at the rate of5.24 mL/kg/hr). At 30 minutes, rivaroxaban infusion was discontinued andr-Antidote administered at 4 mg as an IV bolus over 5 minutes followedby a maintenance infusion of 4 mg/hr for the remainder of the study(additional 55 minutes). Serial blood samples anticoagulated with 3.2%sodium citrate (1:10 dilution) were obtained for measurement of wholeblood INR, rivaroxaban (total and unbound plasma concentrations) andr-Antidote plasma concentrations at 0, 30 (before the end of rivaroxabaninfusion), 35 (end of bolus treatment administration), 60 and 90minutes. Whole blood INR/PT was determined as described in Example 18.r-Antidote concentrations were determined by UNA as described in Example23. Plasma samples were analyzed for rivaroxaban concentration usinghigh-performance-liquid-chromatography with tandem mass spectrometry.Quantitation was performed using a calibration standard curve generatedfrom weighted least square regression analysis. Fraction of rivaroxabannot bound to rat plasma proteins and/or r-antidote protein wasdetermined by an ultrafiltration method using Microcon devices.

FIGS. 29 A and B show sustained reversal of rivaroxaban-inducedanticoagulation by IV administration of r-Antidote in rats as measuredby whole blood INR and PT ratio. The difference in whole blood INR(panel A) between vehicle treated and r-Antidote dosed groups werestatistically significant (p≦0.001) by Student's T test (unpaired twotailed) at 35 and 60 minutes. The difference in PT ratio (panel B)between vehicle treated and r-Antidote dosed groups were statisticallysignificant (p≦0.01) by Student's T test (unpaired two tailed) at 35 and60 minutes. As in Example 19, free (unbound) concentration ofrivaroxaban was greatly reduced upon r-Antidote dosing.

Example 21 Reversal of Activity of LMW Heparin Enoxaparin by r-Antidote

Rats were anesthetized by intraperitoneal dosing of ketamine cocktail,rapidly catheterized for (jugular vein) administration of enoxaparin anda second catheter (femoral vein) was inserted for serial blood sampling.Blood sampling catheter patency was maintained by slow infusion ofnormal saline between samples. Rats were administered enoxaparin(Aventis Pharma, 100 mg/ml) diluted in normal saline at 6, 3, or 1 mg/kgas an IV bolus injection (1 mL). Serial blood samples anticoagulatedwith 3.2% sodium citrate (1:10 dilution) were obtained for measurementof whole blood INR at 0, 2, 15, 30, 60, 90 and 120 minutes postenoxaparin injection. INR measurements were determined using HemochronJr point of care testing device.

As shown in FIG. 30, the three tested doses (1, 3 and 6 mg/kgenoxaparin) produced dose proportional extensions in whole blood INR.Evaluation of anti fXa Units (as measured by Coatest LMW heparin assay)in rat plasma showed that peak anticoagulation corresponded to 4 antifXa U/ml for the 3 mg/kg dose and 1 anti fXa U/ml for the 1 mg/kg dose.As discussed in Example 17, anti-fXa U/ml=1 corresponds to humantherapeutic levels of anticoagulation.

There are no specific reversal agents available for LMW heparins. Thus,protamine sulfate, an agent developed for reversal of activity ofunfractionated heparin during procedures such as coronary artery bypassgraft surgery, is used for this purpose. For enoxaparin, the prescribinginformation describes that neutralization of up to 60% of activity maybe obtained by slow intravenous infusion of protamine sulfate. However,given the incomplete extent of reversal, coupled with the possibility ofhemodynamic and anaphylactic side effects (Weiss and Adkinson, Clin RevAllergy, 1991; 9: 339), this mode of reversal is seldom used as a firstmode of action in enoxaparin treated patients.

In order to test the ability of r-Antidote to reverse enoxaparin inducedanticoagulation and to compare the results to the extent of protaminereversal, we tested the agents in the following regimen: Rats wereadministered enoxaparin diluted into normal saline at 3.0 mg/kg orvehicle (normal saline) as an intravenous bolus injection (1 mL) at t=0.At 10 minutes post enoxaparin injection, vehicle, antidote (5 mg) orprotamine sulfate (0.9 mg, Sigma) was administered intravenously as a 5minute bolus injection. This was followed by a maintenance infusion forthe remainder of the study (additional 45 minutes, 5 mg/h for r-Antidoteand normal saline for protamine). Serial blood samples anticoagulatedwith 3.2% sodium citrate (1:10 dilution) were obtained for measurementof plasma aPTT and antidote concentrations at 0, 5, 15 (end of bolustreatment administration), 30 and 60 minutes. Plasma aPTT measurementswere determined using a MLA Electra 800 Automatic coagulation timer.Calcium chloride and Actin FS PTT reagent (Dade Behring) wereautomatically dispensed to plasma samples (100 uL) per manufacturer'sinstructions.

FIG. 31 shows sustained reversal of enoxaparin-induced anticoagulationupon administration of r-Antidote as well as protamine. The differencein aPTT between vehicle treated and r-Antidote or protamine treatedgroups were statistically significant (p≦0.04) by Student's T test(unpaired two tailed) at 15, 30 and 60 minutes. There was nostatistically significant difference (p=0.27) between the correction ofaPTT by r-Antidote or protamine group. Thus, in this series of ratstudies, r-Antidote could match the anticoagulation reversing ability ofthe currently available antidote for LMW heparin (protamine sulfate).

Example 22 Sustained Reversal of Betrixaban-Induced Anticoagulation byIV Administration of r-Antidote as Measured by Whole Blood INR

Rats were anesthetized and catheterized as described in Example 18. Ratswere administered betrixaban at 1.0 mg/kg/hr IV or vehicle (50%polyethylene glycol in water) for 30 minutes (4.0 mL/kg/hr). At 30minutes, the betrixaban infusion was discontinued and antidoteadministered at 5 mg as an IV bolus over 5 minutes followed by amaintenance infusion of 5 mg/hr for the remainder of the study(additional 55 minutes). Serial blood samples anticoagulated with 3.2%sodium citrate (1:10 dilution) were obtained for measurement of wholeblood INR, betrixaban and antidote concentrations at 0, 30 (before theend of betrixaban infusion), 35 (end of bolus treatment administration),60 and 90 minutes. Whole blood INR measurements were determined on aHemochron Jr device using the citrated PT cartridges. Plasma sampleswere analyzed for betrixaban concentration usinghigh-performance-liquid-chromatography with tandem mass spectrometry.Quantitation was performed using a calibration standard curve generatedfrom weighted least square regression analysis. r-Antidoteconcentrations were determined by ELBA as described in Example 23.

FIG. 32 shows the extent of reversal of betrixaban activity upon dosingof r-antidote. The difference in whole blood INR between vehicle treatedand r-Antidote dosed groups were statistically significant at 60 minutes(p≦0.05) and at 35 and 90 minutes (p≦0.01). Statistical analysis wasperformed by Student's T test (unpaired two tailed). Table 8 shows theratio of r-Antidote to betrixaban required for this sustained correctionof anticoagulation in rats. A twofold ratio of r-Antidote to betrixabanwas required for sustained reversal of anticoagulation in rats.

TABLE 8 Ratio of r-Antidote to betrixaban required for sustainedcorrection in rats Timepoint 30′ 35′ 60′ 90′ Betrixaban alone (uM) 0.1690.062 0.045 0.048 Betrixaban + antidote (uM) 0.154 1.571 1.879 1.190Antidote Concentration (uM) 0.0 5.5 3.5 2.5 Antidote/Betrixaban 0.0 3.51.9 2.1 (molar ratio)

Example 23 Pharmacokinetics of r-Antidote in Rat

One mg of antidote was administered as a short intravenous infusion overfive minutes to four Sprague-Dawley rats. Serial plasma samples werecollected and analyzed for antidote concentration using an enzyme linkedimmunosorption assay (ELISA, Enzyme Research Laboratory, Cat #: FX-EIA).Plasma concentration-time profiles of the antidote was described by atwo compartment model. Systemic clearance of the antidote was low (1.65mL/min/kg) and volume of distribution was small (0.27 L/kg). Thedistribution half-life was 19 minutes followed by a much longer terminalhalf-life of 10 hours. If immediate and near complete reversal ofanticoagulation has to be achieved in a patient, based on ratexperiments (Examples 18, 19, 20 and 22) the ratio of r-Antidoteconcentration to circulating fXa inhibitor concentration is expected tobe targeted at around 2. Thus, to immediately maintain antidote plasmaconcentration above that of a fXa inhibitor, the distribution half-lifeis expected to have a much bigger impact than the terminal half-life onthe human dose selection during an overdose treatment.

FIG. 33 shows plasma concentration-time profile of r-Antidote inSprague-Dawley rats.

Example 24 Pharmacokinetics of r-Antidote in Rhesus Monkey

Two animals were each dosed with 10 mg antidote by intravenous dosingover a ten minute period. Citrate anticoagulated plasma sample analysisfor determination of T_(1/2) were carried out in a manner similar tothat in the rat study (Example 23) except for the pretreatment of plasmasamples with barium citrate absorption to remove endogenous monkey fX.Plasma half life of clearance (T_(1/2)) was approximately 30 minutes(FIG. 34, mean plasma antidote concentration).

In order to remove endogenous fX from monkey plasma to reduceinterference with ELISA measurement, monkey plasma sample (50 uL) wasmixed with 3.2% sodium citrate (5 uL) followed by adding 1 M BaCl2 (5uL). The mixture was kept on ice for 60 min and clarified bycentrifugation with a micro-centrifuge at 13000 rpm for 15 min. Thesupernatant (30 uL) was mixed with 20 uL TBS/EDTA buffer (20 mM Tris/150mM NaCl/50 mM EDTA, pH 7.4). This resulted in 50 uL final mixture with a1:2 dilution of the starting plasma sample. Antidote concentration wasthen determined by the same ELISA procedure as for rat plasma (Example23).

FIG. 34 shows plasma concentration-time profile of r-Antidote in Rhesusmonkeys.

Example 25 Modeling of Projected Human Dose for r-Antidote Reversal offXa Inhibitors

In order to predict human therapeutic doses of r-Antidote, a series ofsimulations were carried out using the WinNonlin software program,version 5.2. Assumptions related to the simulation were as follows:

1. Projected half life of circulation of r-antidote (1 to 3 hours) wasbased on pharmacokinetics in Sprague-Dawley rats (Volume ofdistribution, Vc=13 ml in rats, 3033 ml in human by allometric scaling)(Example 23).2. Plasma concentration of rivaroxaban following 20 mg dose wasextrapolated from literature reports (30 mg dose in healthy elderlyvolunteers, Kubitza D, Becka M, Roth A, Mueck W. Curr Med Res Opin. 2008October; 24(10):2757-65. Epub 2008 Aug. 19). To simulateover-anticoagulation in rivaroxaban treated patients, a twofold higherconcentration was targeted for reversal by r-Antidote.3. Plasma concentration of betrixaban following 40 mg and 80 mg dose wasextrapolated from WO 2008/073670 (which is hereby incorporated byreference in its entirety) and Turpie et al., Thromb Haemost. 2009January; 101(1):68-76. To simulate over-anticoagulation in betrixabantreated patients, a fivefold higher concentration was targeted forreversal by r-Antidote.4. r-Antidote was administered at maximal plasma concentration (C_(max))of rivaroxaban or betrixaban.5. r-Antidote levels were maintained at a 1 to 2-fold higher molarconcentration than that of a fXa inhibitor for a short (1 hour) orextended duration (6 hours). This was to assure near complete reversalof fXa inhibitor anticoagulant activity.6. Pharmacokinetics of r-Antidote followed an one compartment openmodel.

FIGS. 35A and 35B show the simulated time course profile ofneutralization of rivaroxaban activity by administration of r-Antidote.In FIG. 35A, a 20 mg dose of rivaroxaban is reversed by a 400 mg dose ofr-Antidote (bolus dosing) while assuming a T_(1/2) of 3 hours for ther-Antidote. In FIG. 35B, a 20 mg dose of rivaroxaban is reversed using a900 mg dose of r-Antidote (bolus plus 6 hour infusion) while assuming aT_(1/2) of 1 hour for the r-Antidote.

Tables 9-12 shows the projected doses based on the above-notedpredictions.

TABLE 9 Projected doses of r-Antidote needed for reversal of rivaroxaban(10 mg) anticoagulation. Conc. r- Rivaroxaban Antidote Antodote Totalconc. Coverage above T_(1/2) Dose increase time inhibitor (hr) AntidoteDose (mg) (mg) 1x 1 hr 1x 1 IV bolus 60 60 1x 1 hr 1x 3 IV bolus 40 401x 1 hr 2x 1 IV bolus 120 120 1x 1 hr 2x 3 IV bolus 80 80 2x 1 hr 1x 1IV bolus 120 120 2x 1 hr 1x 3 IV bolus 80 80 2x 1 hr 2x 1 IV bolus 240240 2x 1 hr 2x 3 IV bolus 160 160 1x 6 hr 1x 1 Bolus + Infusion  50 +62.5 112.5 or IV bolus 800 800 1x 6 hr 1x 3 IV bolus 50 50 1x 6 hr 2x 1Bolus + Infusion 100 + 125 225 or IV bolus 1600 1600 1x 6 hr 2x 3 IVbolus 100 100 2x 6 hr 1x 1 Bolus + Infusion 100 + 125 225 or IV bolus1600 1600 2x 6 hr 1x 3 IV bolus 100 100 2x 6 hr 2x 1 Bolus + Infusion200 + 250 450 or IV bolus 3200 3200 2x 6 hr 2x 3 IV bolus 200 200

TABLE 10 Projected doses of r-Antidote needed for reversal of betrixaban(80 mg QD) anticoagulation. Conc of Betrixaban Antidote conc. Coverageabove Antodote Total increase time inhibitor T_(1/2) Antidote Dose Dose1x 1 hr 1x 1 IV bolus 20 20 1x 1 hr 1x 3 IV bolus 13 13 1x 1 hr 2x 1 IVbolus 40 40 1x 1 hr 2x 3 IV bolus 26 26 5x 1 hr 1x 1 IV bolus 103 103 5x1 hr 1x 3 IV bolus 66.5 66.5 5x 1 hr 2x 1 IV bolus 206 206 5x 1 hr 2x 3IV bolus 133 133 1x 6 hr 1x 1 Bolus + Infusion 40 + 50 90 or IV bolus190 190 1x 6 hr 1x 3 IV bolus 40 40 1x 6 hr 2x 1 Bolus + Infusion  80 +100 180 or IV bolus 380 380 1x 6 hr 2x 3 IV bolus 80 80 5x 6 hr 1x 1Bolus + Infusion 200 + 250 450 or IV bolus 950 950 5x 6 hr 1x 3 IV bolus200 200 5x 6 hr 2x 1 Bolus + Infusion 400 + 500 900 or IV bolus 19001900 5x 6 hr 2x 3 IV bolus 400 400

TABLE 11 Projected doses of r-Antidote needed for reversal ofrivaroxaban (20 mg) anticoagulation. Conc of Rivaroxaban AntidoteAntodote Total Rivaroxaban Conc Coverage above T½ Dose Dose increasetime inhibitor (hr) Antidote Dose (mg) 20 mg 1x 1 hr 1x 1 IV bolus 120mg 120 1x 1 hr 1x 3 IV bolus 80 mg 80 1x 1 hr 2x 1 IV bolus 240 mg 2401x 1 hr 2x 3 IV bolus 160 mg 160 2x 1 hr 1x 1 IV bolus 240 mg 240 2x 1hr 1x 3 IV bolus 160 mg 160 2x 1 hr 2x 1 IV bolus 480 mg 480 2x 1 hr 2x3 IV bolus 320 mg 320 1x 6 hr 1x 1 Bolus + Infusion 100 + 125 mg 225 orIV bolus 1600 mg 1600 1x 6 hr 1x 3 IV bolus 100 mg 100 1x 6 hr 2x 1Bolus + Infusion 200 + 250 mg 450 1x 6 hr 2x 3 IV bolus 200 mg 200 2x 6hr 1x 1 Bolus + Infusion 200 + 250 mg 450 2x 6 hr 1x 3 IV bolus 100 mg100 2x 6 hr 2x 1 Bolus + Infusion 400 + 500 mg 900 2x 6 hr 2x 3 IV bolus200 mg 200

TABLE 12 Projected doses of r-Antidote needed for reversal of betrixaban(40 mg QD) anticoagulation. Conc of Betrixaban Antidote Antodote TotalBetrixaban Conc Coverage above T½ Dose Dose increase time inhibitor (hr)Antidote Dose (mg) 40 mg QD 1x 1 hr 1x 1 IV bolus 20 mg 20 1x 1 hr 1x 3IV bolus 13 mg 13 1x 1 hr 2x 1 IV bolus 40 mg 40 1x 1 hr 2x 3 IV bolus26 mg 26 5x 1 hr 1x 1 IV bolus 103 mg 103 5x 1 hr 1x 3 IV bolus 66.5 mg66.5 5x 1 hr 2x 1 IV bolus 206 mg 206 5x 1 hr 2x 3 IV bolus 133 mg 1331x 6 hr 1x 1 Bolus + Infusior 40 + 50 mg 90 or IV bolus 190 mg 190 1x 6hr 1x 3 IV bolus 40 mg 40 1x 6 hr 2x 1 Bolus + Infusior 80 + 100 mg 180or IV bolus 380 mg 380 1x 6 hr 2x 3 IV bolus 80 mg 80 0 5x 6 hr 1x 1Bolus + Infusior 200 + 250 mg 450 or IV bolus 950 mg 950 5x 6 hr 1x 3 IVbolus 200 mg 200 5x 6 hr 2x 1 Bolus + Infusior 400 + 500 mg 900 or IVbolus 1900 mg 1900 5x 6 hr 2x 3 IV bolus 400 mg 400

Example 26 Modeling of Projected Human Dose for r-Antidote Reversal ofLMW Heparins

In order to predict human therapeutic doses of r-Antidote, a series ofsimulations were carried out using the following assumptions:

1. According to enoxaparin prescribing information, peak anti fXaactivity in unstable angina patients treated with 1 mg/kg enoxaparin bysubcutaneous dosing correspond to 1.1 U/ml. In order to mimicsupratherapeutic levels of anticoagulation, a circulating anti fXa unitrange of 2-4 U/ml was targeted for reversal.2. Mean absolute bioavailability of enoxaparin by subcutaneous dosing is92% in healthy human volunteers. Therefore results from rat intravenousdosing studies were assumed to be equivalent to those obtained bysubcutaneous dosing.3 Projected human doses to reverse anticoagulant effect of LMW heparinswas calculated from the effective dose in rat and scaled to human bycorrecting for the difference in blood volume between the species (B.Davies and T Morris, Pharm Res, 10 (7), 1993, pp 1093-1095).4. Measurements of anti-fXa units for LMW heparins in rat plasma wereconsidered to be equivalent to those measured in human plasma.5. Complete reversal of pharmacodynamic marker (aPTT or anti fXa units)was necessary for neutralization of fXa inhibitor activity andrestoration of hemostatic capability (i.e., complete reversal ofanticoagulation).Results of the simulation showed that:A) Total dose of r-Antidote for reversal of activity of therapeuticlevels of enoxaparin was between 500 mg and 1 gB) Total dose of r-Antidote for reversal of activity of supratherapeuticlevels of enoxaparin was between 500 mg and 2 g.

FIGS. 36A and 36B show the simulated time course profile ofneutralization of betrixaban activity by r-Antidote. In FIG. 36A, a 80mg dose of betrixaban is reversed by a 400 mg dose of r-Antidote (bolusdosing) while assuming a T_(1/2) of 3 hours for the r-Antidote. In FIG.36B a 80 mg dose of betrixaban is reversed using a 900 mg dose ofr-Antidote (bolus plus 6 hour infusion) while assuming a T_(1/2) of 1hour for the r-Antidote.

Example 27 Reversal of Rivaroxaban in Rhesus Monkey by r-Antidote

In FIG. 37, the effect of r-Antidote on reversal of anticoagulation byrivaroxaban is provided based on testing in citrate anticoagulatedplasma from four rhesus monkeys. Prothrombin times were measured as inExample 12. Addition of rivaroxaban (250 nM or 1 uM) produceddose-responsive prolongation of prothrombin times (PT) over baselineclotting times of individual monkeys. Addition of 250 nM rivaroxabanproduced an extension to 32.3±6.1 sec (average±standard deviation) froma baseline value of 17.5±1.6 sec. Addition of r-Antidote to therivaroxaban treated plasma sample reversed the anticoagulant effect with244 nM correcting PT to 25±7 sec and 488 nM correcting PT to 19.9±1.9sec. Addition of r-Antidote alone to baseline plasma did not change PT(17.7 sec).

Example 28 Reversal of Blood Loss Due to Enoxaparin and Fondaparinux inRat by r-Antidote

The effect of r-Antidote on reversal of blood loss due to enoxaparinanticoagulation was tested in Sprague-Dawley rats. Specifically, rattail transection blood loss model for restoration of hemostasis wasemployed. The enoxaparin was dosed by IV bolus (4.5 mg/kg). Ther-Antidote was administered as two doses: 1) a 2 milligram bolus andthen infused at a rate of 2 mg/hour for a total of 15 minutes; and 2) a4 milligram bolus and then infused at a rate of 4 mg/hour for a total of15 minutes. Blood loss reversal was also tested using the vehicle.Immediately after the bolus injection of the r-Antidote was complete andthe infusion started, the tip of the rat's tail was transected with ascalpel blade and placed into a vial containing physiological saline at37° C. The tail was allowed to bleed for 15 minutes. The resulting bloodvolume was determined by lysis of red blood cells, measuring thehemoglobin concentration spectrophotometrically and estimating the bloodvolume by comparison against a standard curve. Decreased blood losscorrelated with both r-Antidote plasma concentrations (r²=0.80) and areduction of anti-fXa units (r²=0.89). The results are provided in FIG.38 (AD refers to the r-Antidote).

In the same model, r-Antidote completely corrected increased blood lossdue to fondaparinux (25 mg/kg) administration. Protamine (provided as a0.9 mg IV bolus) failed to display corrective activity. The r-Antidotewas provided as a 6 mg bolus and then another 6 g/hr for 15 min as aninfusion. The results are provided in FIG. 39.

As can be seen from FIG. 38 and FIG. 39, these results demonstrate that,in addition to neutralizing direct fXa inhibitors, r-Antidote is alsocapable of neutralizing indirect fXa inhibitors and has the potential torestore hemostasis by reversal of anticoagulation medicated by bothclasses of drugs.

In the same model, blood loss due to enoxaparin (dosed as a 4.5 mg/kg IVbolus) was reduced to 42% by r-Antidote administration at 2 mg bolusfollowed by a 2 mg/hr infusion and completely reversed by r-Antidoteadministration at 4 mg bolus followed by a 4 mg/hr infusion. Theseresults are depicted in FIG. 41.

In the same model, blood loss due to fondaparinux (provided as a 25mg/kg IV bolus) was completely reversed by r-Antidote administration at6 mg bolus followed by a 6 mg/hr infusion. In contrast, protamineadministered as a 0.9 mg bolus did not reverse blood loss. These resultsare depicted in FIG. 43.

This data is consistent with the currently claimed invention based onthe assumption that the rat weighs approximately 200 times that of ahuman.

Example 29 Reversal of Enoxaparin- and Fondaparinux-InducedAnticoagulation after Bolus r-Antidote Administration as Measured byPlasma Anti-fXa Units

Anti-fXa activity has been used in clinics for measuring anticoagulationlevels obtained with LMWH treatment and correlated with clinicaloutcomes (Montalescot et al., Circulation, 2004, 110(4):392-8).

The effect of r-Antidote on enoxaparin-induced anticoagulation wasmeasured by plasma anti-fXa activity assay. The anti-fXa activity assayis based on a modified LMWH assay kit (Coamatic LMWH) which expressesthe anticoagulant activity of LMWH in terms of anti-fXa units. Theanti-fXa units assay measures residual fXa (bovine fXa) activity inplasma using a fXa chromogenic substrate (S2732). Known concentrationsof enoxaparin standard (U/ml) were used to construct a standard curvefor measurement of anti-fXa units (U/ml) in unknown samples.

The r-Antidote was administered as a bolus at a dose of 1 mg, 2 mg, or 4mg. These results are depicted in FIG. 40. As can be seen, the antidotereversed the enoxaparin-induced anticoagulation in a dose-responsivemanner.

In the same model, fondaparinux was administrated at a dose of 1 mg/kg(IV bolus) followed by r-Antidote (4 mg bolus starting at 5 minutes+4mg/hr infusion for the duration of the experiment). As shown in FIG. 44,the increase in anti-fXa activity due to fondaparinux was rapidly andsubstantially reversed by the administration of r-Antidote.

The anti-fXa activity for fondaparinux was expressed as μg/m by using aknown concentration of fondaparinux as a standard. When measured usingenoxaparin as the standard, 1 μg/m fondaparinux was equivalent to 0.66U/ml enoxaparin in rat plasma, or 0.80 U/ml enoxaparin in human plasma.

Example 30 Correlation of Blood Loss, Anti-fXa Unit, and rfXa AntidoteConcentrations in Rat Tail Transection Model

FIGS. 42 a, 42 b, and 42 c show the correlation between blood loss inthe rat tail transection model and enoxaparin concentrations as measuredby anti-fXa units (r²=0.887). The anti-fXa units and rfXa antidoteconcentration refer to the levels obtained at the 15 minute timepoint—just prior to tail transection. The results demonstrated a steepincrease in blood loss as enoxaparin concentrations increased to >1.5anti-fXa Units/ml with a plateau in the maximum blood loss achieved withthis model representing approximately 5% of normal rat blood volume lostover the 15 minute collection time. Higher doses of enoxaparin weretested during initial model development experiments but no greater bloodloss was demonstrated. Further correlation analysis showed an r²=0.887between blood loss and rfXa antidote concentrations and r²=0.689 betweenanti-fXa units and rfXa antidote concentrations.

It is to be understood that while the invention has been described inconjunction with the above embodiments, that the foregoing descriptionand examples are intended to illustrate and not limit the scope of theinvention. Other aspects, advantages and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

TABLE 13 Sequence ID NO. 1-Polypeptide Sequence of Human Factor X 1MGRPLHLVLL SASLAGLLLL GESLFIRREQ ANNILARVTR ANSFLEEMKK GHLERECMEE 61TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK CKDGLGEYTC TCLEGFEGKN 121CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN GKACIPTGPY PCGKQTLERR 181KRSVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF NQTQPERGDN NLTRIVGGQE 241CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ AKRFKVRVGD RNTEQEEGGE 301AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP ACLPERDWAE STLMTQKTGI 361VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ NMFCAGYDTK QEDACQGDSG 421GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK WIDRSMKTRG LPKAKSHAPE 481VITSSPLK

TABLE 14 Sequence ID NO. 2-A polynucleotide Sequence Encoding Factor X 1gactttgctc cagcagcctg tcccagtgag gacagggaca cagtactcgg ccacaccatg 61gggcgcccac tgcacctcgt cctgctcagt gcctccctgg ctggcctcct gctgctcggg 121gaaagtctgt tcatccgcag ggagcaggcc aacaacatcc tggcgagggt cacgagggcc 181aattcctttc ttgaagagat gaagaaagga cacctcgaaa gagagtgcat ggaagagacc 241tgctcatacg aagaggcccg cgaggtcttt gaggacagcg acaagacgaa tgaattctgg 301aataaataca aagatggcga ccagtgtgag accagtcctt gccagaacca gggcaaatgt 361aaagacggcc tcggggaata cacctgcacc tgtttagaag gattcgaagg caaaaactgt 421gaattattca cacggaagct ctgcagcctg gacaacgggg actgtgacca gttctgccac 481gaggaacaga actctgtggt gtgctcctgc gcccgcgggt acaccctggc tgacaacggc 541aaggcctgca ttcccacagg gccctacccc tgtgggaaac agaccctgga acgcaggaag 601aggtcagtgg cccaggccac cagcagcagc ggggaggccc ctgacagcat cacatggaag 661ccatatgatg cagccgacct ggaccccacc gagaacccct tcgacctgct tgacttcaac 721cagacgcagc ctgagagggg cgacaacaac ctcaccagga tcgtgggagg ccaggaatgc 781aaggacgggg agtgtccctg gcaggccctg ctcatcaatg aggaaaacga gggtttctgt 841ggtggaacca ttctgagcga gttctacatc ctaacggcag cccactgtct ctaccaagcc 901aagagattca aggtgagggt aggggaccgg aacacggagc aggaggaggg cggtgaggcg 961gtgcacgagg tggaggtggt catcaagcac aaccggttca caaaggagac ctatgacttc 1021gacatcgccg tgctccggct caagaccccc atcaccttcc gcatgaacgt ggcgcctgcc 1081tgcctccccg agcgtgactg ggccgagtcc acgctgatga cgcagaagac ggggattgtg 1141agcggcttcg ggcgcaccca cgagaagggc cggcagtcca ccaggctcaa gatgctggag 1201gtgccctacg tggaccgcaa cagctgcaag ctgtccagca gcttcatcat cacccagaac 1261atgttctgtg ccggctacga caccaagcag gaggatgcct gccaggggga cagcgggggc 1321ccgcacgtca cccgcttcaa ggacacctac ttcgtgacag gcatcgtcag ctggggagag 1381ggctgtgccc gtaaggggaa gtacgggatc tacaccaagg tcaccgcctt cctcaagtgg 1441atcgacaggt ccatgaaaac caggggcttg cccaaggcca agagccatgc cccggaggtc 1501ataacgtcct ctccattaaa gtgagatccc actcaaaaaa aaaaaaaaaa aaaaaaaaaa

TABLE 15 Sequence ID NO. 3-Polypeptide Sequence of Mature Human Factor X1 ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLERR KRSVAQATSS SGEAPDSITW KPYDAADLDP TENPFDLLDF 181NQTQPERGDN NLTRIVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 16 Sequence ID NO. 4-Polypeptide Sequence of the Gla-domainless Factor Xa lacking 1 to 44 amino acid residues Light Chain 1    KDGDQC ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFCHEEQNSVVCS CARGYTLADN 121 GKACIPTGPY PCGKQTLER Heavy Chain 181                IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 17 Sequence ID NO. 5-Polypeptide Sequence of the Gla-domainless Factor Xa lacking 1 to 45 amino acid residues Light Chain 1     DGDQC ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFCHEEQNSVVCS CARGYTLADN 121 GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 18 Sequence ID NO. 6-Polypeptide Sequence ofActivated Human Factor Xa prior to Post-Translation of Glutamic Acid to γ- Carboxyglutamic acid Light Chain 1ANSFLEEMKK GHLERECMEE TCSYEEAREV FEDSDKTNEF WNKYKDGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 19 Sequence ID NO. 7-Polypeptide Sequence ofActivated Human Factor Xa with Post- Translation of Glutamic Acid to γ-Carboxyglutamic acid (γ represents γ-Carboxyglutamic Acid Residue)Light Chain 1 ANSFLγγMKK GHLγRγCMγγ TCSYγγARyV FγDSDKTNγFWNKYKDGDQC ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFCHEEQNSVVCS CARGYTLADN 121 GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 20 Sequence ID NO. 8-Polypeptide Sequence ofActivated Human Factor Xa-Light Chain withPost-Translation of Glutamic Acid to γ- Carboxyglutamic acid Light Chain1 ANSFLγγMKK GHLγRγCMγγ TCSYγγARγV FγDSDKTNγF WNKYKDGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER

TABLE 21 Sequence ID NO. 9-Polypeptide Sequence ofActivated Human Factor Xa-Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDSG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 22 Sequence ID NO. 10-Polypeptide Sequence of theDes-Gla Anhydro Factor Xa (Ã represents dehydroalanine) Light Chain 1    KDGDQC ETSPCQNQGK 61 CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFCHEEQNSVVCS CARGYTLADN 121 GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDÃG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 23 Sequence ID NO. 11-Polypeptide Sequence ofthe Des-Gla fXa-S379A Light Chain 1      DGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 24 Sequence ID NO. 12-Polypeptide Sequence of a HumanFactor Xa triple mutant prior to removal of theRKRRKR (SEQ ID NO. 17) linker Light Chain 1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Linker RKRRKR Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 25 Sequence ID NO. 13-Polypeptide Sequence of a HumanFactor Xa triple mutant after removal of theRKRRKR (SEQ ID NO. 17) linker Light Chain 1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER Heavy Chain 181               IVGGQE CKDGECPWQA LLINEENEGF CGGTILSEFY ILTAAHCLYQ 241AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYD FDIAVLRLKT PITFRMNVAP 301ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKML EVPYVDRNSC KLSSSFIITQ 361NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWG EGCARKGKYG IYTKVTAFLK 421WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 26 Sequence ID NO. 14-Polypeptide Sequence of LightChain Fragment of Human Factor Xa triple mutant after secretion 1ANSFL                                     F WNKYKDGDQC ETSPCQNQGK 61CKDGLGEYTC TCLEGFEGKN CELFTRKLCS LDNGDCDQFC HEEQNSVVCS CARGYTLADN 121GKACIPTGPY PCGKQTLER

TABLE 27 Sequence ID NO. 15-Polypeptide Sequence of HeavyChain Fragment of Human Factor Xa triple mutant after secretionHeavy Chain 181                IVGGQE CKDGECPWQA LLINEENEGFCGGTILSEFY ILTAAHCLYQ 241 AKRFKVRVGD RNTEQEEGGE AVHEVEVVIK HNRFTKETYDFDIAVLRLKT PITFRMNVAP 301 ACLPERDWAE STLMTQKTGI VSGFGRTHEK GRQSTRLKMLEVPYVDRNSC KLSSSFIITQ 361 NMFCAGYDTK QEDACQGDAG GPHVTRFKDT YFVTGIVSWGEGCARKGKYG IYTKVTAFLK 421 WIDRSMKTRG LPKAKSHAPE VITSSPLK

TABLE 28 Sequence ID NO. 16-A polynucleotide SequenceEncoding r-Antidote (a Factor X triple mutant) 1ATGGGGCGCC CACTGCACCT CGTCCTGCTC AGTGCCTCCCTGGCTGGCCT CCTGCTGCTC GGGGAAAGTC TGTTCATCCG CAGGGAGCAG GCCAACAACA 101TCCTGGCGAG GGTCACGAGG GCCAATTCCT TTCTTTTCTGGAATAAATAC AAAGATGGCG ACCAGTGTGA GACCAGTCCT TGCCAGAACC AGGGCAAATG 201TAAAGACGGC CTCGGGGAAT ACACCTGCAC CTGTTTAGAAGGATTCGAAG GCAAAAACTG TGAATTATTC ACACGGAAGC TCTGCAGCCT GGACAACGGG 301GACTGTGACC AGTTCTGCCA CGAGGAACAG AACTCTGTGGTGTGCTCCTG CGCCCGCGGG TACACCCTGG CTGACAACGG CAAGGCCTGC ATTCCCACAG 401GGCCCTACCC CTGTGGGAAA CAGACCCTGG AACGCAGGAAGAGGAGGAAG AGGATCGTGG GAGGCCAGGA ATGCAAGGAC GGGGAGTGTC CCTGGCAGGC 501CCTGCTCATC AATGAGGAAA ACGAGGGTTT CTGTGGTGGAACCATTCTGA GCGAGTTCTA CATCCTAACG GCAGCCCACT GTCTCTACCA AGCCAAGAGA 601TTCAAGGTGA GGGTAGGGGA CCGGAACACG GAGCAGGAGGAGGGCGGTGA GGCGGTGCAC GAGGTGGAGG TGGTCATCAA GCACAACCGG TTCACAAAGG 701AGACCTATGA CTTCGACATC GCCGTGCTCC GGCTCAAGACCCCCATCACC TTCCGCATGA ACGTGGCGCC TGCCTGCCTC CCCGAGCGTG ACTGGGCCGA 801GTCCACGCTG ATGACGCAGA AGACGGGGAT TGTGAGCGGCTTCGGGCGCA CCCACGAGAA GGGCCGGCAG TCCACCAGGC TCAAGATGCT GGAGGTGCCC 901TACGTGGACC GCAACAGCTG CAAGCTGTCC AGCAGCTTCATCATCACCCA GAACATGTTC TGTGCCGGCT ACGACACCAA GCAGGAGGAT GCCTGCCAGG 1001GGGACGCAGG GGGCCCGCAC GTCACCCGCT TCAAGGACACCTACTTCGTG ACAGGCATCG TCAGCTGGGG AGAGGGCTGT GCCCGTAAGG GGAAGTACGG 1101GATCTACACC AAGGTCACCG CCTTCCTCAA GTGGATCGACAGGTCCATGA AAACCAGGGG CTTGCCCAAG GCCAAGAGCC ATGCCCCGGA GGTCATAACG 1201TCCTCTCCAT TAAAGTGA

TABLE 29 Sequence ID. NO. 18-Polynucleotide Sequence of ther-Antidote Expression Vector    1TCTAGACACA GTACTCGGCC ACACCATGGG GCGCCCACTGCACCTCGTCC TGCTCAGTGC CTCCCTGGCT GGCCTCCTGC TGCTCGGGGA AAGTCTGTTC  101ATCCGCAGGG AGCAGGCCAA CAACATCCTG GCGAGGGTCACGAGGGCCAA TTCCTTTCTT TTCTGGAATA AATACAAAGA TGGCGACCAG TGTGAGACCA  201GTCCTTGCCA GAACCAGGGC AAATGTAAAG ACGGCCTCGGGGAATACACC TGCACCTGTT TAGAAGGATT CGAAGGCAAA AACTGTGAAT TATTCACACG  301GAAGCTCTGC AGCCTGGACA ACGGGGACTG TGACCAGTTCTGCCACGAGG AACAGAACTC TGTGGTGTGC TCCTGCGCCC GCGGGTACAC CCTGGCTGAC  401AACGGCAAGG CCTGCATTCC CACAGGGCCC TACCCCTGTGGGAAACAGAC CCTGGAACGC AGGAAGAGGA GGAAGAGGAT CGTGGGAGGC CAGGAATGCA  501AGGACGGGGA GTGTCCCTGG CAGGCCCTGC TCATCAATGAGGAAAACGAG GGTTTCTGTG GTGGAACCAT TCTGAGCGAG TTCTACATCC TAACGGCAGC  601CCACTGTCTC TACCAAGCCA AGAGATTCAA GGTGAGGGTAGGGGACCGGA ACACGGAGCA GGAGGAGGGC GGTGAGGCGG TGCACGAGGT GGAGGTGGTC  701ATCAAGCACA ACCGGTTCAC AAAGGAGACC TATGACTTCGACATCGCCGT GCTCCGGCTC AAGACCCCCA TCACCTTCCG CATGAACGTG GCGCCTGCCT  801GCCTCCCCGA GCGTGACTGG GCCGAGTCCA CGCTGATGACGCAGAAGACG GGGATTGTGA GCGGCTTCGG GCGCACCCAC GAGAAGGGCC GGCAGTCCAC  901CAGGCTCAAG ATGCTGGAGG TGCCCTACGT GGACCGCAACAGCTGCAAGC TGTCCAGCAG CTTCATCATC ACCCAGAACA TGTTCTGTGC CGGCTACGAC 1001ACCAAGCAGG AGGATGCCTG CCAGGGGGAC GCAGGGGGCCCGCACGTCAC CCGCTTCAAG GACACCTACT TCGTGACAGG CATCGTCAGC TGGGGAGAGG 1101GCTGTGCCCG TAAGGGGAAG TACGGGATCT ACACCAAGGTCACCGCCTTC CTCAAGTGGA TCGACAGGTC CATGAAAACC AGGGGCTTGC CCAAGGCCAA 1201GAGCCATGCC CCGGAGGTCA TAACGTCCTC TCCATTAAAGTGAGATCCCA CTCGGATCCC TATTCTATAG TGTCACCTAA ATGCTAGAGC TCGCTGATCA 1301GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTTGCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT 1401AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCATTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG 1501CAGGCATGCT GGGGATGCGG TGGGCTCTAT GGCTTCTGAGGCGGAAAGAA CCAGCTGGGG CTCGAGCGGC CGCCCCTTCT GAGGCGGAAA GAACCAGCTG 1601TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCTCCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC AGGTGTGGAA 1701AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCATGCATCTCAAT TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC 1801TCCGCCCAGT TCCGCCCATT CTCCGCCCCA TGGCTGACTAATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCT CTGAGCTATT CCAGAAGTAG 1901TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT TGCAAAAAAGCTAGCTTCCC GCTGCCATCA TGGTTCGACC ATTGAACTGC ATCGTCGCCG TGTCCCAAAA 2001TATGGGGATT GGCAAGAACG GAGACCTACC CTGGCCTCCGCTCAGGAACG AGTTCAAGTA CTTCCAAAGA ATGACCACAA CCTCTTCAGT GGAAGGTAAA 2101CAGAATCTGG TGATTATGGG TAGGAAAACC TGGTTCTCCATTCCTGAGAA GAATCGACCT TTAAAGGACA GAATTAATAT AGTTCTCAGT AGAGAACTCA 2201AAGAACCACC ACGAGGAGCT CATTTTCTTG CCAAAAGTTTGGATGATGCC TTAAGACTTA TTGAACAACC GGAATTGGCA AGTAAAGTAG ACATGGTTTG 2301GATAGTCGGA GGCAGTTCTG TTTACCAGGA AGCCATGAATCAACCAGGCC ACCTTAGACT CTTTGTGACA AGGATCATGC AGGAATTTGA AAGTGACACG 2401TTTTTCCCAG AAATTGATTT GGGGAAATAT AAACTTCTCCCAGAATACCC AGGCGTCCTC TCTGAGGTCC AGGAGGAAAA AGGCATCAAG TATAAGTTTG 2501AAGTCTACGA GAAGAAAGAC TAACAGGAAG ATGCTTTCAAGTTCTCTGCT CCCCTCCTAA AGCTATGCAT TTTTATAAGA CCATGGGACT TTTGCTGGCT 2601TTAGATCCCG CGGAGATCCA GACATGATAA GATACATTGATGAGTTTGGA CAAACCACAA CTAGAATGCA GTGAAAAAAA TGCTTTATTT GTGAAATTTG 2701TGATGCTATT GCTTTATTTG TAACCATTAT AAGCTGCAATAAACAAGTTA ACAACAACAA TTGCATTCAT TTTATGTTTC AGGTTCAGGG GGAGGTGTGG 2801GAGGTTTTTT AAAGCAAGTA AAACCTCTAC AAATGTGGTATGGCTGATTA TGAGCTCCAG CTTTTGTTCC CTTTAGTGAG GGTTAATTGC GCGCTTGGCG 2901TAATCATGGT CATAGCTGTT TCCTGTGTGA AATTGTTATCCGCTCACAAT TCCACACAAC ATACGAGCCG GAAGCATAAA GTGTAAAGCC TGGGGTGCCT 3001AATGAGTGAG CTAACTCACA TTAATTGCGT TGCGCTCACTGCCCGCTTTC CAGTCGGGAA ACCTGTCGTG CCAGCTGCAT TAATGAATCG GCCAACGCGC 3101GGGGAGAGGC GGTTTGCGTA TTGGGCGCTC TTCCGCTTCCTCGCTCACTG ACTCGCTGCG CTCGGTCGTT CGGCTGCGGC GAGCGGTATC AGCTCACTCA 3201AAGGCGGTAA TACGGTTATC CACAGAATCA GGGGATAACGCAGGAAAGAA CATGTGAGCA AAAGGCCAGC AAAAGGCCAG GAACCGTAAA AAGGCCGCGT 3301TGCTGGCGTT TTTCCATAGG CTCCGCCCCC CTGACGAGCATCACAAAAAT CGACGCTCAA GTCAGAGGTG GCGAAACCCG ACAGGACTAT AAAGATACCA 3401GGCGTTTCCC CCTGGAAGCT CCCTCGTGCG CTCTCCTGTTCCGACCCTGC CGCTTACCGG ATACCTGTCC GCCTTTCTCC CTTCGGGAAG CGTGGCGCTT 3501TCTCATAGCT CACGCTGTAG GTATCTCAGT TCGGTGTAGGTCGTTCGCTC CAAGCTGGGC TGTGTGCACG AACCCCCCGT TCAGCCCGAC CGCTGCGCCT 3601TATCCGGTAA CTATCGTCTT GAGTCCAACC CGGTAAGACACGACTTATCG CCACTGGCAG CAGCCACTGG TAACAGGATT AGCAGAGCGA GGTATGTAGG 3701CGGTGCTACA GAGTTCTTGA AGTGGTGGCC TAACTACGGCTACACTAGAA GGACAGTATT TGGTATCTGC GCTCTGCTGA AGCCAGTTAC CTTCGGAAAA 3801AGAGTTGGTA GCTCTTGATC CGGCAAACAA ACCACCGCTGGTAGCGGTGG TTTTTTTGTT TGCAAGCAGC AGATTACGCG CAGAAAAAAA GGATCTCAAG 3901AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTGGAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA TCAAAAAGGA TCTTCACCTA 4001GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAAAGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC 4101TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGACTCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA 4201TGATACCGCG AGACCCACGC TCACCGGCTC CAGATTTATCAGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA CTTTATCCGC 4301CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTAAGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG CATCGTGGTG 4401TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTTCCCAACGATC AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT 4501TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGTGTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC CATCCGTAAG 4601ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTCTGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAATACG GGATAATACC 4701GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAAAACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT 4801AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTACTTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA AATGCCGCAA AAAAGGGAAT 4901AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTTTTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA 5001TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACATTTCCCCGAAA AGTGCCACCT GGGAAATTGT AAACGTTAAT ATTTTGTTAA AATTCGCGTT 5101AAATTTTTGT TAAATCAGCT CATTTTTTAA CCAATAGGCCGAAATCGGCA AAATCCCTTA TAAATCAAAA GAATAGACCG AGATAGGGTT GAGTGTTGTT 5201CCAGTTTGGA ACAAGAGTCC ACTATTAAAG AACGTGGACTCCAACGTCAA AGGGCGAAAA ACCGTCTATC AGGGCGATGG CCCACTACGT GAACCATCAC 5301CCTAATCAAG TTTTTTGGGG TCGAGGTGCC GTAAAGCACTAAATCGGAAC CCTAAAGGGA GCCCCCGATT TAGAGCTTGA CGGGGAAAGC CGGCGAACGT 5401GGCGAGAAAG GAAGGGAAGA AAGCGAAAGG AGCGGGCGCTAGGGCGCTGG CAAGTGTAGC GGTCACGCTG CGCGTAACCA CCACACCCGC CGCGCTTAAT 5501GCGCCGCTAC AGGGCGCGTC GCGCCATTCG CCATTCAGGCTGCGCAACTG TTGGGAAGGG CGATCGGTGC GGGCCTCTTC GCTATTACGC CAGCTGGCGA 5601AAGGGGGATG TGCTGCAAGG CGATTAAGTT GGGTAACGCCAGGGTTTTCC CAGTCACGAC GTTGTAAAAC GACGGCCAGT GAGCGCGCGT AATACGACTC 5701ACTATAGGGC GAATTGGAAT TAATTCGCTG GGCTGAGACCCGCAGAGGAA GACGCTCTAG GGATTTGTCC CGGACTAGCG AGATGGCAAG GCTGAGGACG 5801GGAGGCTGAT TGAGAGGCGA AGGTACACCC TAATCTCAATACAACCCTTG GAGCTAAGCC AGCAATGGTA GAGGGAAGAT TCTGCACGTC CCTTCCAGGC 5901GGCCTCCCCG TCACCACCCA CCCCAACCCG CCCCGACCGGAGCTGAGAGT AATTCATACA AAAGGACTCG CCCCTGCCTT GGGGAATCCC AGGGACCGTC 6001GTTAAACTCC CACTAACGTA GAACCCAGAG ATCGCTGCGTTCCCGCCCCC TCACCCGCCC GCTCTCGTCA TCACTGAGGT GGAGAAGAGC ATGCGTGAGG 6101CTCCGGTGCC CGTCAGTGGG CAGAGCGCAC ATCGCCCACAGTCCCCGAGA AGTTGGGGGG AGGGGTCGGC AATTGAACCG GTGCCTAGAG AAGGTGGCGC 6201GGGGTAAACT GGGAAAGTGA TGTCGTGTAC TGGCTCCGCCTTTTTCCCGA GGGTGGGGGA GAACCGTATA TAAGTGCAGT AGTCGCCGTG AACGTTCTTT 6301TTCGCAACGG GTTTGCCGCC AGAACACAGG TAAGTGCCGTGTGTGGTTCC CGCGGGCCTG GCCTCTTTAC GGGTTATGGC CCTTGCGTGC CTTGAATTAC 6401TTCCACGCCC CTGGCTGCAG TACGTGATTC TTGATCCCGAGCTTCGGGTT GAAAGTGGGT GGGAGAGTTC GAGGCCTTGC GCTTAAGGAG CCCCTTCGCC 6501TCGTGCTTGA GTTGAGGCCT GGCTTGGGCG CTGGGGCCGCCGCGTGCGAA TCTGGTGGCA CCTTCGCGCC TATCTCGCTG CTTTCGATAA GTCTCTAGCC 6601ATTTAAAATT TTTGATGACC TGCTGCGACG CTTTTTTTCTGGCAAGATAG TCTTGTAAAT GCGGGCCAAG ATCTGCACAC TGGTATTTCG GTTTTTGGGG 6701CCGCGGGCGG CGACGGGGCC CGTGCGTCCC AGCGCACATGTTCGGCGAGG CGGGGCCTGC GAGCGCGGCC ACCGAGAATC GGACGGGGGT AGTCTCAAGC 6801TGGCCGGCCT GCTCTGGTGC CTGGCCTCGC GCCGCCGTGTATCGCCCCGC CCTGGGCGGC AAGGCTGGCC CGGTCGGCAC CAGTTGCGTG AGCGGAAAGA 6901TGGCCGCTTC CCGGCCCTGC TGCAGGGAGC TCAAAATGGAGGACGCGGCG CTCGGGAGAG CGGGCGGGTG AGTCACCCAC ACAAAGGAAA AGGGCCTTTC 7001CGTCCTCAGC CGTCGCTTCA TGTGACTCCA CGGAGTACCGGGCGCCGTCC AGGCACCTCG ATTAGTTCTC GAGCTTTTGG AGTACGTCGT CTTTAGGTTG 7101GGGGGAGGGG TTTTATGCGA TGGAGTTTCC CCACACTGAGTGGGTGGAGA CTGAAGTTAG GCCAGCTTGG CACTTGATGT AATTCTCCTT GGAATTTGCC 7201CTTTTTGAGT TTGGATCTTG GTTCATTCTC AAGCCTCAGACAGTGGTTCA AAGTTTTTTT CTTCCATTTC AGGTGTCGTG AAAACTACCC CTAAAAGCCA 7301AAT

1. A unit dose formulation comprising a pharmaceutically acceptablecarrier and from about 200 milligrams to about 1 gram of a two chainpolypeptide comprising the amino acid sequence of SEQ ID NO.
 13. 2-3.(canceled)
 4. The unit dose formulation of claim 1, having from about400 milligrams to about 900 milligrams of the polypeptide. 5-20.(canceled)
 21. A method of preventing, reducing, or ceasing bleeding ina subject undergoing anticoagulant therapy with a factor Xa inhibitorcomprising administering to the subject a unit dose formulation ofclaim
 1. 22-24. (canceled)
 25. The method of claim 21, wherein the unitdose formulation is administered intravenously by bolus.
 26. The methodof claim 21, wherein the unit dose formulation is administered as aninfusion or a combination of bolus plus infusion.
 27. The method ofclaim 26, wherein about 10 to about 20% of the formulation isadministered as a bolus and the remaining formulation is infused over aperiod until bleeding has substantially ceased.
 28. The method of claim26, wherein the formulation is administered for about 6 hours.
 29. Themethod of claim 26, wherein the formulation is administered for about 6to about 12 hours.
 30. The method of claim 26, wherein the formulationis administered for about 12 to about 24 hours.
 31. The method of claim26, wherein the formulation is administered for up to about 48 hours.32. A package or kit comprising a container containing a unit doseformulation comprising from about 200 milligrams to about 1 gram of atwo-chain polypeptide comprising the amino acid sequence of SEQ ID NO.13 and a package insert indicating that the unit dose formulation issuitable for preventing, reducing or ceasing bleeding in a human subjectundergoing an anticoagulant therapy with a factor Xa (fXa) inhibitor,whether the fXa inhibitor is a direct fXa inhibitor or an indirect fXainhibitor.